Antibodies directed against trail

ABSTRACT

A novel cytokine designated TRAIL induces apoptosis of certain target cells, including cancer cells and virally infected cells. Isolated DNA sequences encoding TRAIL are disclosed, along with expression vectors and transformed host cells useful in producing TRAIL polypeptides. Antibodies that specifically bind TRAIL are provided as well.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/320,424,filed May 26, 1999, now U.S. Pat. No. 6,284,236, which is acontinuation-in-part of application Ser. No. 09/190,046, filed Nov. 10,1998, now abandoned, which is a continuation-in-part of application Ser.No. 09/048,641, filed Mar. 26, 1998, now abandoned, which is acontinuation-in-part of application Ser. No. 08/670,354, filed Jun. 25,1996, now U.S. Pat. No. 5,763,223, which is a continuation-in-part ofapplication Ser. No. 08/548,368, filed Nov. 1, 1995, now abandoned,which is a continuation-in-part of application Ser. No. 08/496,632,filed Jun. 29, 1995, now abandoned.

BACKGROUND OF THE INVENTION

The programmed cell death known as apoptosis is distinct from cell deathdue to necrosis. Apoptosis occurs in embryogenesis, metamorphosis,endocrine-dependent tissue atrophy, normal tissue turnover, and death ofimmune thymocytes (induced through their antigen-receptor complex or byglucocorticoids) (Itoh et al., Cell 66:233, 1991). During maturation ofT-cells in the thymus, T-cells that recognize self-antigens aredestroyed through the apoptotic process, whereas others are positivelyselected. The possibility that some T-cells recognizing certain selfepitopes (e.g., inefficiently processed and presented antigenicdeterminants of a given self protein) escape this elimination processand subsequently play a role in autoimmune diseases has been suggested(Gammon et al., Immunology Today 12:193, 1991).

A cell surface antigen known as Fas has been reported to mediateapoptosis and is believed to play a role in clonal deletion ofself-reactive T-cells (Itoh et al., Cell 66:233, 1991; Watanabe-Fukunageet al., Nature 356:314, 1992). Cross-linking a specific monoclonalantibody to Fas has been reported to induce various cell lines toundergo apoptosis (Yonehara et al., J. Exp. Med., 169:1747, 1989; Trauthet al., Science, 245:301, 1989). However, under certain conditions,binding of a specific monoclonal antibody to Fas can have acostimulatory effect on freshly isolated T cells (Alderson et al., J.Exp. Med. 178:2231, 1993).

DNAs encoding a rat Fas ligand (Suda et al., Cell, 75:1169, 1993) and ahuman Fas ligand (Takahashi et al., International Immunology 6:1567,1994) have been isolated. Binding of the Fas ligand to cells expressingFas antigen has been demonstrated to induce apoptosis (Suda et al.,supra, and Takahashi et al., supra).

Investigation into the existence and identity of other molecule(s) thatplay a role in apoptosis is desirable. Identifying such molecules wouldprovide an additional means of regulating apoptosis, as well asproviding further insight into the development of self-tolerance by theimmune system and the etiology of autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides a novel cytokine protein, as well asisolated DNA encoding the cytokine and expression vectors comprising theisolated DNA. Properties of the novel cytokine, which is a member of thetumor necrosis factor (TNF) family of ligands, include the ability toinduce apoptosis of certain types of target cells. This protein thus isdesignated TNF Related Apoptosis Inducing Ligand (TRAIL). Among thetypes of cells that are killed by contact with TRAIL are cancer cellssuch as leukemia, lymphoma, and melanoma cells, and cells infected witha virus.

A method for producing TRAIL polypeptides involves culturing host cellstransformed with a recombinant expression vector that containsTRAIL-encoding DNA under conditions appropriate for expression of TRAIL,then recovering the expressed TRAIL polypeptide from the culture.Antibodies directed against TRAIL polypeptides are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of an assay described in example 8. Theassay demonstrated that a soluble human TRAIL polypeptide induced deathof Jurkat cells, which are a leukemia cell line.

FIG. 2 presents the results of an assay described in example 11. Contactwith a soluble human TRAIL polypeptide induced death ofcytomegalovirus-infected human fibroblasts, whereas non-virally infectedfibroblasts were not killed.

FIG. 3 depicts a particular fusion protein encoded by an expressionvector of the present invention. The fusion protein comprises (from N-to C-terminus) a growth hormone-derived leader sequence (SEQ ID NO:19),followed by a tripeptide encoded by an oligonucleotide employed invector construction, a leucine zipper peptide (SEQ ID NO:15), atripeptide encoded by an oligonucleotide employed in vectorconstruction, and a soluble human TRAIL polypeptide (amino acids 95 to281 of SEQ ID NO:2). A DNA sequence encoding the fusion protein, and theamino acid sequence of the fusion protein, are presented in SEQ ID NO:10and 11, respectively.

FIG. 4 depicts a fusion protein encoded by another expression vector ofthe present invention, comprising (from N- to C-terminus) acytomegalovirus-derived leader sequence (amino acids 1 to 29 of SEQ IDNO:9), followed by a tripeptide encoded by an oligonucleotide employedin vector construction (amino acids 30 to 32 of SEQ ID NO:9), a leucinezipper peptide (SEQ ID NO:15), a tripeptide encoded by anoligonucleotide employed in vector construction, and a soluble humanTRAIL polypeptide (amino acids 95 to 281 of SEQ ID NO:2). A DNA sequenceencoding the fusion protein, and the amino acid sequence of the fusionprotein, are presented in SEQ ID NO:12 and 13, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A novel protein designated TRAIL is provided herein, along with DNAencoding TRAIL and recombinant expression vectors comprising TRAIL DNA.A method for producing recombinant TRAIL polypeptides involvescultivating host cells transformed with the recombinant expressionvectors under conditions appropriate for expression of TRAIL, andrecovering the expressed TRAIL.

The present invention also provides antibodies that specifically bindTRAIL proteins. In one embodiment, the antibodies are monoclonalantibodies.

The TRAIL protein induces apoptosis of certain types of target cells,such as transformed cells that include but are not limited to cancercells and virally-infected cells. As demonstrated in examples 5, 8, 9,and 10 below, TRAIL induced apoptosis of human leukemia, lymphoma, andmelanoma cell lines. Among the uses of TRAIL is use in killing cancercells. TRAIL finds further use in treatment of viral infections.Infection with cytomegalovirus (CMV) rendered human fibroblastssusceptible to apoptosis when contacted with TRAIL, whereas uninfectedfibroblasts were not killed through contact with TRAIL (see example 11).

Isolation of a DNA encoding human TRAIL is described in example 1 below.The nucleotide sequence of the human TRAIL DNA isolated in example 1 ispresented in SEQ ID NO:1, and the amino acid sequence encoded thereby ispresented in SEQ ID NO:2. This human TRAIL protein comprises anN-terminal cytoplasmic domain (amino acids 1-18), a transmembrane region(amino acids 19-38), and an extracellular domain (amino acids 39-281).The extracellular domain contains a receptor-binding region.

E. coli strain DH10B cells transformed with a recombinant vectorcontaining this human TRAIL DNA were deposited with the American TypeCulture Collection on Jun. 14, 1995, and assigned accession no. 69849.The deposit was made under the terms of the Budapest Treaty. Therecombinant vector in the deposited strain is the expression vectorpDC409 (described in example 5). The vector was digested with SalI andNotI, and human TRAIL DNA that includes the entire coding region shownin SEQ ID NO:1 was ligated into the vector.

DNA encoding a second human TRAIL protein was isolated as described inexample 2. The nucleotide sequence of this DNA is presented in SEQ IDNO:3, and the amino acid sequence encoded thereby is presented in SEQ IDNO:4. The encoded protein comprises an N-terminal cytoplasmic domain(amino acids 1-18), a transmembrane region (amino acids 19-38), and anextracellular domain (amino acids 39-101).

The DNA of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID NO:1, and isthus designated the human TRAIL deletion variant (huTRAILdv) clone.Nucleotides 18 through 358 of SEQ ID NO:1 are identical to nucleotides 8through 348 of the huTRAILdv DNA of SEQ ID NO:3. Nucleotides 359 through506 of SEQ ID NO:1 are missing from the cloned DNA of SEQ ID NO:3. Thedeletion causes a shift in the reading frame, which results in anin-frame stop codon after amino acid 101 of SEQ ID NO:4. The DNA of SEQID NO:3 thus encodes a truncated protein. Amino acids 1 through 90 ofSEQ ID NO:2 are identical to amino acids 1 through 90 of SEQ ID NO:4.However, due to the deletion, the C-terminal portion of the huTRAILdvprotein (amino acids 91 through 101 of SEQ ID NO:4) differs from theresidues in the corresponding positions in SEQ ID NO:2. In contrast tothe full length huTRAIL protein, the truncated huTRAILdv protein doesnot exhibit the ability to induce apoptosis of the T cell leukemia cellsof the Jurkat cell line.

DNA encoding a mouse TRAIL protein has also been isolated, as describedin example 3. The nucleotide sequence of this DNA is presented in SEQ IDNO:5 and the amino acid sequence encoded thereby is presented in SEQ IDNO:6. The encoded protein comprises an N-terminal cytoplasmic domain(amino acids 1-17), a transmembrane region (amino acids 18-38), and anextracellular domain (amino acids 39-291). This mouse TRAIL is 64%identical to the human TRAIL of SEQ ID NO:2 at the amino acid level. Thecoding region of the mouse TRAIL nucleotide sequence is 75% identical tothe coding region of the human nucleotide sequence of SEQ ID NO:1.

One embodiment of the present invention is directed to human TRAILprotein characterized by the N-terminal amino acid sequenceMetAlaMetMetGluValGlnGly GlyProSerLeuGlyGlnThr (amino acids 1-15 of SEQID NOS:2 and 4). Mouse TRAIL proteins characterized by the N-terminalamino acid sequence MetProSerSerGlyAla LeuLysAspLeuSerPheSerGlnHis(amino acids 1-15 of SEQ ID NO:6) are also provided herein.

The TRAIL of the present invention is distinct from the protein known asFas ligand (Suda et al., Cell, 75:1169, 1993; Takahashi et al.,International Immunology 6:1567, 1994). Fas ligand induces apoptosis ofcertain cell types, via the receptor known as Fas. As demonstrated inexample 5, TRAIL-induced apoptosis of target cells is not mediatedthrough Fas. The human TRAIL amino acid sequence of SEQ ID NO:2 is about20% identical to the human Fas ligand amino acid sequence that ispresented in Takahashi et al., supra. The extracellular domain of humanTRAIL is about 28.4% identical to the extracellular domain of human Fasligand.

The amino acid sequences disclosed herein reveal that TRAIL is a memberof the TNF family of ligands (Smith et al. Cell, 73:1349, 1993; Suda etal., Cell, 75:1169, 1993; Smith et al., Cell, 76:959, 1994). The percentidentities between the human TRAIL extracellular domain amino acidsequence and the amino acid sequence of the extracellular domain ofother proteins of this family are as follows: 28.4% with Fas ligand,22.4% with lymphotoxin-β, 22.9% with TNF-α, 23.1% with TNF-β, 22.1% withCD30 ligand, and 23.4% with CD40 ligand.

TRAIL was tested for ability to bind receptors of the TNF-R family ofreceptors. The binding analysis was conducted using the slideautoradiography procedure of Gearing et al. (EMBO J. 8:3667, 1989). Theanalysis revealed no detectable binding of human TRAIL to human CD30,CD40, 4-1BB, OX40, TNF-R (p80 form), CD27, or LTβR (also known asTNFR-RP). The results in example 5 indicate that human TRAIL does notbind human Fas.

The TRAIL polypeptides of the present invention include polypeptideshaving amino acid sequences that differ from, but are highly homologousto, those presented in SEQ ID NOS:2 and 6. Examples include, but are notlimited to, homologs derived from other mammalian species, variants(both naturally occurring variants and those generated by recombinantDNA technology), and TRAIL fragments that retain a desired biologicalactivity. Such polypeptides exhibit a biological activity of the TRAILproteins of SEQ ID NOS:2 and 6, and preferably comprise an amino acidsequence that is at least 80% identical (most preferably at least 90%identical) to the amino acid sequence presented in SEQ ID NO:2 or SEQ IDNO:6. These embodiments of the present invention are described in moredetail below.

Conserved sequences located in the C-terminal portion of proteins in theTNF family are identified in Smith et al. (Cell, 73:1349, 1993, see page1353 and FIG. 6); Suda et al. (Cell, 75:1169, 1993, see FIG. 7); Smithet al. (Cell, 76:959, 1994, see FIG. 3); and Goodwin et al. (Eur. J.Immunol., 23:2631, 1993, see FIG. 7 and pages 2638-39), herebyincorporated by reference. Among the amino acids in the human TRAILprotein that are conserved (in at least a majority of TNF familymembers) are those in positions 124-125 (AH), 136 (L), 154 (W), 169 (L),174 (L), 180 (G), 182 (Y), 187 (Q), 190 (F), 193 (Q), and 275-276 (FG)of SEQ ID NO:2. Another structural feature of TRAIL is a spacer regionbetween the C-terminus of the trans-membrane region and the portion ofthe extracellular domain that is believed to be most important forbiological activity. This spacer region, located at the N-terminus ofthe extracellular domain, consists of amino acids 39 through 94 of SEQID NO:2. Analogous spacers are found in other family members, e.g., CD40ligand. Amino acids 138 through 153 correspond to a loop between the βsheets of the folded (three dimensional) human TRAIL protein.

Provided herein are membrane-bound TRAIL proteins (comprising acytoplasmic domain, a transmembrane region, and an extracellular domain)as well as TRAIL fragments that retain a desired biological property ofthe full length TRAIL protein. In one embodiment, TRAIL fragments aresoluble TRAIL polypeptides comprising all or part of the extracellulardomain, but lacking the transmembrane region that would cause retentionof the polypeptide on a cell membrane. Soluble TRAIL proteins arecapable of being secreted from the cells in which they are expressed.Advantageously, a heterologous signal peptide is fused to the N-terminussuch that the soluble TRAIL is secreted upon expression.

Soluble TRAIL may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired protein from the culture medium, e.g., by centrifugation,and assaying the medium (supernatant) for the presence of the desiredprotein. The presence of TRAIL in the medium indicates that the proteinwas secreted from the cells and thus is a soluble form of the TRAILprotein. Naturally-occurring soluble forms of TRAIL are encompassed bythe present invention.

The use of soluble forms of TRAIL is advantageous for certainapplications. Purification of the proteins from recombinant host cellsis facilitated, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

Examples of soluble TRAIL polypeptides are those containing the entireextracellular domain (e.g., amino acids 39 to 281 of SEQ ID NO:2 oramino acids 39 to 291 of SEQ ID NO:6). Fragments of the extracellulardomain that retain a desired biological activity are also provided. Suchfragments advantageously include regions of TRAIL that are conserved inproteins of the TNF family of ligands, as described above.

Additional examples of soluble TRAIL polypeptides are those lacking notonly the cytoplasmic domain and transmembrane region, but also all orpart of the above-described spacer region. Soluble human TRAILpolypeptides thus include, but are not limited to, polypeptidescomprising amino acids x to 281, wherein x represents any of the aminoacids in positions 39 through 95 of SEQ ID NO:2. In the embodiment inwhich residue 95 is the N-terminal amino acid, the entire spacer regionhas been deleted.

TRAIL fragments, including soluble polypeptides, may be prepared by anyof a number of conventional techniques. A DNA sequence encoding adesired TRAIL fragment may be subcloned into an expression vector forproduction of the TRAIL fragment. The TRAIL-encoding DNA sequenceadvantageously is fused to a sequence encoding a suitable leader orsignal peptide. The desired TRAIL-encoding DNA fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

The well known polymerase chain reaction (PCR) procedure also may beemployed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to faciliate insertion of the amplified DNA fragment intoan expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

As will be understood by the skilled artisan, the transmembrane regionof each TRAIL protein discussed above is identified in accordance withconventional criteria for identifying that type of hydrophobic domain.The exact boundaries of a transmembrane region may vary slightly (mostlikely by no more than five amino acids on either end) from thosepresented above. Computer programs useful for identifying suchhydrophobic regions in proteins are available.

The TRAIL DNA of the present invention includes cDNA, chemicallysynthesized DNA, DNA isolated by PCR, genomic DNA, and combinationsthereof. Genomic TRAIL DNA may be isolated by hybridization to the TRAILcDNA disclosed herein using standard techniques. RNA transcribed fromthe TRAIL DNA is also encompassed by the present invention.

A search of the NCBI databank identified five expressed sequence tags(ESTs) having regions of identity with TRAIL DNA. These ESTs (NCBIaccession numbers T90422, T82085, T10524, R31020, and Z36726) are allhuman cDNA fragments. The NCBI records do not disclose any polypeptideencoded by the ESTs, and do not indicate what the reading frame, if any,might be. However, even if the knowledge of the reading frame revealedherein by disclosure of complete TRAIL coding regions is used to expressthe ESTs, none of the encoded polypeptides would have theapoptosis-inducing property of the presently-claimed TRAIL polypeptides.In other words, if each of the five ESTs were inserted into expressionvectors downstream from an initiator methionine codon, in the readingframe elucidated herein, none of the resulting expressed polypeptideswould contain a sufficient portion of the extracellular domain of TRAILto induce apoptosis of Jurkat cells.

Certain embodiments of the present invention provide isolated DNAcomprising a nucleotide sequence selected from the group consisting ofnucleotides 88 to 933 of SEQ ID NO:1 (human TRAIL coding region);nucleotides 202 to 933 of SEQ ID NO:1 (encoding the human TRAILextracellular domain); nucleotides 47 to 922 of SEQ ID NO:5 (mouse TRAILcoding region); and nucleotides 261 to 922 of SEQ ID NO:5 (encoding themouse TRAIL extracellular domain). DNAs encoding biologically activefragments of the proteins of SEQ ID NOS:2 and 6 are also provided.Further embodiments include sequences comprising nucleotides 370 to 930of SEQ ID NO:1 and nucleotides 341 to 919 of SEQ ID NO:5, which encodethe particular human and murine soluble TRAIL polypeptides,respectively, described in example 7.

Due to degeneracy of the genetic code, two DNA sequences may differ, yetencode the same amino acid sequence. The present invention thus providesisolated DNA sequences encoding biologically active TRAIL, selected fromDNA comprising the coding region of a native human or murine TRAIL CDNA,or fragments thereof, and DNA which is degenerate as a result of thegenetic code to the native TRAIL DNA sequence.

Also provided herein are purified TRAIL polypeptides, both recombinantand non-recombinant. Variants and derivatives of native TRAIL proteinsthat retain a desired biological activity are also within the scope ofthe present invention. In one embodiment, the biological activity of anTRAIL variant is essentially equivalent to the biological activity of anative TRAIL protein. One desired biological activity of TRAIL is theability to induce death of Jurkat cells. Assay procedures for detectingapoptosis of target cells are well known. DNA laddering is among thecharacteristics of cell death via apoptosis, and is recognized as one ofthe observable phenomena that distinguish apoptotic cell death fromnecrotic cell death. Examples of assay techniques suitable for detectingdeath or apoptosis of target cells include those described in examples 5and 8 to 11. Another property of TRAIL is the ability to bind to Jurkatcells.

TRAIL variants may be obtained by mutations of native TRAIL nucleotidesequences, for example. A TRAIL variant, as referred to herein, is apolypeptide substantially homologous to a native TRAIL, but which has anamino acid sequence different from that of native TRAIL because of oneor a plurality of deletions, insertions or substitutions. TRAIL-encodingDNA sequences of the present invention encompass sequences that compriseone or more additions, deletions, or substitutions of nucleotides whencompared to a native TRAIL DNA sequence, but that encode an TRAILprotein that is essentially biologically equivalent to a native TRAILprotein.

The variant amino acid or DNA sequence preferably is at least 80%identical to a native TRAIL sequence, most preferably at least 90%identical. The degree of homology (percent identity) between a nativeand a mutant sequence may be determined, for example, by comparing thetwo sequences using computer programs commonly employed for thispurpose. One suitable program is the GAP computer program, version 6.0,described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). Briefly, the GAP program defines identityas the number of aligned symbols (i.e., nucleotides or amino acids)which are identical, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Techniques for making such alterations include those disclosedby Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, Jan. 12-19, 1985); Smith et al. (GeneticEngineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat.Nos. 4,518,584 and 4,737,462, which are incorporated by referenceherein.

Variants may comprise conservatively substituted sequences, meaning thatone or more amino acid residues of a native TRAIL polypeptide arereplaced by different residues, but that the conservatively substitutedTRAIL polypeptide retains a desired biological activity that isessentially equivalent to that of a native TRAIL polypeptide. Examplesof conservative substitutions include substitution of amino acids thatdo not alter the secondary and/or tertiary structure of TRAIL. Otherexamples involve substitution of amino acids outside of thereceptor-binding domain, when the desired biological activity is theability to bind to a receptor on target cells and induce apoptosis ofthe target cells. A given amino acid may be replaced by a residue havingsimilar physiochemical characteristics, e.g., substituting one aliphaticresidue for another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. TRAIL polypeptidescomprising conservative amino acid substitutions may be tested in one ofthe assays described herein to confirm that a desired biologicalactivity of a native TRAIL is retained. DNA sequences encoding TRAILpolypeptides that contain such conservative amino acid substitutions areencompassed by the present invention.

Conserved amino acids located in the C-terminal portion of proteins inthe TNF family, and believed to be important for biological activity,have been identified. These conserved sequences are discussed in Smithet al. (Cell, 73:1349, 1993, see page 1353 and FIG. 6); Suda et al.(Cell, 75:1169, 1993, see FIG. 7); Smith et al. (Cell, 76:959, 1994, seeFIG. 3); and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993, see FIG. 7and pages 2638-39). Advantageously, the conserved amino acids are notaltered when generating conservatively substituted sequences. Ifaltered, amino acids found at equivalent positions in other members ofthe TNF family are substituted.

TRAIL also may be modified to create TRAIL derivatives by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of TRAIL may be prepared by linking the chemical moieties tofunctional groups on TRAIL amino acid side chains or at the N-terminusor C-terminus of a TRAIL polypeptide or the extracellular domainthereof. Other derivatives of TRAIL within the scope of this inventioninclude covalent or aggregative conjugates of TRAIL or its fragmentswith other proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatemay comprise a signal or leader polypeptide sequence (e.g. the α-factorleader of Saccharomyces) at the N-terminus of a TRAIL polypeptide. Thesignal or leader peptide co-translationally or post-translationallydirects transfer of the conjugate from its site of synthesis to a siteinside or outside of the cell membrane or cell wall.

TRAIL polypeptide fusions can comprise peptides added to facilitatepurification and identification of TRAIL. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDK) (SEQ ID NO:7), which is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, thusenabling rapid assay and facile purification of expressed recombinantprotein. This sequence is also specifically cleaved by bovine mucosalenterokinase at the residue immediately following the Asp-Lys pairing.Fusion proteins capped with this peptide may also be resistant tointracellular degradation in E. coli.

A murine hybridoma designated 4E11 produces a monoclonal antibody thatbinds the peptide DYKDDDDK (SEQ ID NO:7) in the presence of certaindivalent metal cations (as described in U.S. Pat. No. 5,011,912), andhas been deposited with the American Type Culture Collection underaccession no HB 9259. Expression systems useful for producingrecombinant proteins fused to the FLAG® peptide, as well as monoclonalantibodies that bind the peptide and are useful in purifying therecombinant proteins, are available from Eastman Kodak Company,Scientific Imaging Systems, New Haven, Conn.

The present invention further includes TRAIL polypeptides with orwithout associated native-pattern glycosylation. TRAIL expressed inyeast or mammalian expression systems may be similar to or significantlydifferent from a native TRAIL polypeptide in molecular weight andglycosylation pattern, depending upon the choice of expression system.Expression of TRAIL polypeptides in bacterial expression systems, suchas E. coli, provides non-glycosylated molecules.

Glycosylation sites in the TRAIL extracellular domain can be modified topreclude glycosylation while allowing expression of a homogeneous,reduced carbohydrate analog using yeast or mammalian expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Yis Ser or Thr. Appropriate modifications to the nucleotide sequenceencoding this triplet will result in substitutions, additions ordeletions that prevent attachment of carbohydrate residues at the Asnside chain. Known procedures for inactivating N-glycosylation sites inproteins include those described in U.S. Pat. No. 5,071,972 and EP276,846. A potential N-glycosylation site is found at positions 109-111in the human protein of SEQ ID NO:2 and at positions 52-54 in the murineprotein of SEQ ID NO:6.

Alternatively, known procedures such as mutagenesis may be employed toadd glycosylation sites to TRAIL, thereby promoting an increase in thecarbohydrate moieties attached to TRAIL. Such an approach may be takenwhen slowing the clearance of TRAIL from the body following in vivoadministration is desired, for example.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Cysteine residues are found in the human TRAIL protein ofSEQ ID NO:2 at positions 16, 30, 56, 77, and 230; and in the murineTRAIL protein of SEQ ID NO:6 at positions 22, 60, 81, and 240.

Among the soluble human TRAIL polypeptides disclosed herein arefragments of the extracellular domain that lack the spacer region, asdescribed above. Such spacer-deleted soluble TRAIL polypeptides includeonly one cysteine, corresponding to the residue at position 230 of SEQID NO:2. Thus, any disulfide bonds forming from the Cys-230 residuewould be intermolecular, joining two such soluble TRAIL polypeptides. Inthe fusion protein of FIG. 3 (SEQ ID NO:11), the TRAIL polypeptidemoiety comprises only one cysteine, at position 202 (which correspondsto the cysteine residue at position 230 in the full length human TRAILsequence of SEQ ID NO:2). In the fusion protein of FIG. 4 (SEQ IDNO:13), the TRAIL polypeptide comprises only one cysteine, at position205 (which corresponds to the Cys-230 residue in SEQ ID NO:2).

One embodiment of the invention is directed to a TRAIL polypeptide (orfusion protein comprising a TRAIL polypeptide), in which the cysteineresidue corresponding to the cysteine at position 230 in SEQ ID NO:2 isdeleted or substituted, in order to prevent formation of disulfide bondsthat involve the Cys-230 residue. If substituted, cysteine may bereplaced by any suitable amino acid, whereby a desired biologicalactivity of TRAIL is maintained. Examples include, but are not limitedto, serine, alanine, glycine, or valine. Altering the number of cysteineresidues to manipulate oligomer formation is discussed further below.

Other variants are prepared by modification of adjacent dibasic aminoacid residues to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.Potential KEX2 protease processing sites are found at positions 89-90and 149-150 in the protein of SEQ ID NO:2, and at positions 85-86,135-136, and 162-163 in the protein of SEQ ID NO:6.

Naturally occurring TRAIL variants are also encompassed by the presentinvention. Examples of such variants are proteins that result fromalternative mRNA splicing events (since TRAIL is encoded by a multi-exongene) or from proteolytic cleavage of the TRAIL protein, wherein adesired biological activity is retained. Alternative splicing of MRNAmay yield a truncated but biologically active TRAIL protein, such as anaturally occurring soluble form of the protein, for example. Variationsattributable to proteolysis include, for example, differences in the N-or C-termini upon expression in different types of host cells, due toproteolytic removal of one or more terminal amino acids from the TRAILprotein. In addition, proteolytic cleavage may release a soluble form ofTRAIL from a membrane-bound form of the protein. Allelic variants arealso encompassed by the present invention.

Also provided herein are conjugates or fusion proteins comprising aTRAIL polypeptide and a tumor-targeting moiety. Such embodiments may beemployed in cancer treatment, for example. The TRAIL component may beany of the various forms of TRAIL disclosed herein, with one examplebeing a soluble TRAIL polypeptide. Oligomers comprising such fusionproteins also are contemplated. The conjugates or fusion proteins mayadditionally comprise other components of TRAIL-containing fusions,compositions, and the like that are described herein. Examples of suchother components include, but are not limited to, leucine zipperpeptides.

The tumor-targeting moiety may be any compound that enhances delivery ofTRAIL to a tumor. Such compounds include, but are not limited to,compounds that selectively bind to cancer cells compared with normalcells, specifically bind to a particular type of cancer that is to betreated, or enhance penetration into solid tumors. In one embodiment,the tumor-targeting moiety is a peptide.

Examples of tumor-targeting peptides are described in Arap et al.(Science 279:377, Jan. 16, 1998), and Pasqualini et al. (NatureBiotechnology 15:542, June 1997), which are hereby incorporated byreference in their entirety. Arap et al. and Pasqualini et al. reportstudies of peptides that “home” to tumors, selectively binding to tumorvessels and/or to tumor cells. The tripeptides Arg-Gly-Asp, Asn-Gly-Argand Gly-Ser-Leu, or peptides comprising such tripeptide sequences, arecontemplated herein for use as tumor targeting moieties as components ofTRAIL fusion proteins.

Arap et al. and Pasqualini et al., supra, disclose that peptidescomprising the sequence Arg-Gly-Asp (RGD) bind to integrins, includingbut not limited to α_(v) integrins. Such integrins have been detected intumor vasculature and on a number of tumor cell types.

Arap et al. additionally disclose that peptides comprising thetripeptide Asn-Gly-Arg or Gly-Ser-Leu selectively bind to tumors. Amongthe peptides studied by Arap et al. are the RGD-containing peptideCDCRGDCFC (SEQ ID NO:20), and the NGR-containing peptides CNGRCVSGCAGRC(SEQ ID NO:21), NGRAHA (SEQ ID NO:22), CVLNGRMEC (SEQ ID NO:23), andCNGRC (SEQ ID NO:24).

One type of fusion protein provided herein comprises a TRAIL polypeptideand a peptide that binds an integrin associated with a tumor. Suchintegrin may be expressed on tumor cells or tumor vessels, for example.An example of an integrin is an α_(v) integrin. Arap et al., supra, notethat human α_(v) integrins are selectively expressed in human tumorblood vessels.

Oligomers

The present invention encompasses TRAIL polypeptides in the form ofoligomers, such as dimers, trimers, or higher oligomers. Oligomers maybe formed by disulfide bonds between cysteine residues on differentTRAIL polypeptides, or by non-covalent interactions between TRAILpolypeptide chains, for example. In other embodiments, oligomerscomprise from two to four TRAIL polypeptides joined via covalent ornon-covalent interactions between peptide moieties fused to the TRAILpolypeptides. Such peptides may be peptide linkers (spacers), orpeptides that have the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of TRAIL polypeptides attachedthereto, as described in more detail below. The TRAIL polypeptidespreferably are soluble.

Preparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991); Byrn et al. (Nature 344:667, 1990); and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992),hereby incorporated by reference. In one embodiment of the invention, anTRAIL dimer is created by fusing TRAIL to an Fc region polypeptidederived from an antibody. The term “Fc polypeptide” includes native andmutein forms, as well as truncated Fc polypeptides containing the hingeregion that promotes dimerization. The Fc polypeptide preferably isfused to a soluble TRAIL (e.g., comprising only the extracellulardomain).

A gene fusion encoding the TRAIL/Fc fusion protein is inserted into anappropriate expression vector. In one embodiment, the Fc polypeptide isfused to the N-terminus of a soluble TRAIL polypeptide. The TRAIL/Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between the Fc polypeptides,yielding divalent TRAIL. In other embodiments, TRAIL may be substitutedfor the variable portion of an antibody heavy or light chain. If fusionproteins are made with both heavy and light chains of an antibody, it ispossible to form an TRAIL oligomer with as many as four TRAILextracellular regions.

One suitable Fc polypeptide is the native Fc region polypeptide derivedfrom a human IgG1, which is described in PCT application WO 93/10151,hereby incorporated by reference. Another useful Fc polypeptide is theFc mutein described in U.S. Pat. No. 5,457,035. The amino acid sequenceof the mutein is identical to that of the native Fc sequence presentedin WO 93/10151, except that amino acid 19 has been changed from Leu toAla, amino acid 20 has been changed from Leu to Glu, and amino acid 22has been changed from Gly to Ala. This mutein Fc exhibits reducedaffinity for immunoglobulin receptors.

Alternatively, oligomeric TRAIL may comprise two or more soluble TRAILpolypeptides joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Fusion proteins comprising multiple TRAILpolypeptides separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing oligomeric TRAIL polypeptides involves useof a leucine zipper. Leucine zipper domains are peptides that promoteoligomerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988), and have since been found in a varietyof different proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.

Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, 1988). Zipper domain is aterm used to refer to a conserved peptide domain present in these (andother) proteins, which is responsible for oligomerization of theproteins. The zipper domain (also referred to herein as anoligomerizing, or oligomer-forming, domain) comprises a repetitiveheptad repeat, often with four or five leucine residues interspersedwith other amino acids. Examples of zipper domains are those found inthe yeast transcription factor GCN4 and a heat-stable DNA-bindingprotein found in rat liver (C/EBP; Landschulz et al., Science 243:1681,1989). Two nuclear transforming proteins, fos and jun, also exhibitzipper domains, as does the gene product of the murine proto-oncogene,c-myc (Landschulz et al., Science 240:1759, 1988). The products of thenuclear oncogenes fos and jun comprise zipper domains preferentiallyform a heterodimer (O'Shea et al., Science 245:646, 1989; Turner andTjian, Science 243:1689, 1989). The zipper domain is necessary forbiological activity (DNA binding) in these proteins.

The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton,Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and HumanRetroviruses 6:703, 1990). The zipper domains in these fusogenic viralproteins are near the transmembrane region of the proteins; it has beensuggested that the zipper domains could contribute to the oligomericstructure of the fusogenic proteins. Oligomerization of fusogenic viralproteins is involved in fusion pore formation (Spruce et al, Proc. Natl.Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been recentlyreported to play a role in oligomerization of heat-shock transcriptionfactors (Rabindran et al., Science 259:230, 1993).

Zipper domains fold as short, parallel coiled coils. (O'Shea et al.,Science 254:539; 1991). The general architecture of the parallel coiledcoil has been well characterized, with a “knobs-into-holes” packing asproposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed bya zipper domain is stabilized by the heptad repeat, designated(abcdefg)_(n) according to the notation of McLachlan and Stewart (J.Mol. Biol. 98:293; 1975), in which residues a and d are generallyhydrophobic residues, with d being a leucine, which line up on the sameface of a helix. Oppositely-charged residues commonly occur at positionsg and e. Thus, in a parallel coiled coil formed from two helical zipperdomains, the “knobs” formed by the hydrophobic side chains of the firsthelix are packed into the “holes” formed between the side chains of thesecond helix.

The residues at position d (often leucine) contribute large hydrophobicstabilization energies, and are important for oligomer formation(Krystek et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al.recently reported the synthesis of a triple-stranded α-helical bundle inwhich the helices run up-up-down (Science 259:1288, 1993). Their studiesconfirmed that hydrophobic stabilization energy provides the maindriving force for the formation of coiled coils from helical monomers.These studies also indicate that electrostatic interactions contributeto the stoichiometry and geometry of coiled coils. Further discussion ofthe structure of leucine zippers is found in Harbury et al. (Science262:1401, Nov. 26, 1993).

Several studies have indicated that conservative amino acids may besubstituted for individual leucine residues with minimal decrease in theability to dimerize; multiple changes, however, usually result in lossof this ability (Landschulz et al., Science 243:1681, 1989; Turner andTjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). vanHeekeren et al. reported that a number of different amino residues canbe substituted for the leucine residues in the zipper domain of GCN4,and further found that some GCN4 proteins containing two leucinesubstitutions were weakly active (Nucl. Acids Res. 20:3721, 1992).Mutation of the first and second heptadic leucines of the zipper domainof the measles virus fusion protein (MVF) did not affect syncytiumformation (a measure of virally-induced cell fusion); however, mutationof all four leucine residues prevented fusion completely (Buckland etal., J. Gen. Virol. 73:1703, 1992). None of the mutations affected theability of MVF to form a tetramer.

Examples of leucine zipper domains suitable for producing solubleoligomeric TRAIL proteins include, but are not limited to, thosedescribed in PCT application WO 94/10308 and in U.S. Pat. No. 5,716,805,hereby incorporated by reference. Recombinant fusion proteins comprisinga soluble TRAIL polypeptide, fused to a peptide that dimerizes ortrimerizes in solution, are expressed in suitable host cells, and theresulting soluble oligomeric TRAIL is recovered from the culturesupernatant. DNA encoding such fusion proteins is provided herein.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,Cell 73:431, 1993). Thus, trimeric TRAIL may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties arethose that preferentially form trimers. One example is a leucine zipperderived from lung surfactant protein D (SPD), as described in Hoppe etal. (FEBS Letters 344:191, 1994) and in U.S. Pat. No. 5,716,805, herebyincorporated by reference in their entirety. This lung SPD-derivedleucine zipper peptide comprises the amino acid sequence Pro Asp Val AlaSer Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln AlaAla Phe Ser Gln Tyr (SEQ ID NO:14).

Another example of a leucine zipper that promotes trimerization is apeptide comprising the amino acid sequence Arg Met Lys Gln Ile Glu AspLys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala ArgIle Lys Lys Leu Ile Gly Glu Arg (residues 2 to 34 of SEQ ID NO:15), asdescribed in U.S. Pat. No. 5,716,805. In an alternative embodiment, thepeptide lacks the N-terminal Arg residue, thus comprising residues 3 to34 of SEQ ID NO:15. In another embodiment, an N-terminal Asp residue isadded, such that the peptide comprises the sequence Asp Arg Met Lys GlnIle Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn GluIle Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg (SEQ ID NO:15).

Yet another example of a suitable leucine zipper peptide comprises theamino acid sequence Ser Leu Ala Ser Leu Arg Gln Gln Leu Glu Ala Leu GlnGly Gln Leu Gln His Leu Gln Ala Ala Leu Ser Gln Leu Gly Glu (SEQ IDNO:16). In an alternative peptide, the leucine residues in the foregoingsequence are replaced with isoleucine: Ser Ile Ala Ser Ile Arg Gln GlnIle Glu Ala Ile Gln Gly Gln Ile Gln His Ile Gln Ala Ala Ile Ser Gln IleGly Glu (SEQ ID NO:17). Fragments of the foregoing zipper peptides thatretain the property of promoting oligomerization may be employed aswell. Examples of such fragments include, but are not limited to,peptides lacking one or two of the N-terminal or C-terminal residuespresented in the foregoing amino acid sequences.

Other peptides derived from naturally occurring trimeric proteins may beemployed in preparing trimeric TRAIL. Alternatively, synthetic peptidesthat promote oligomerization may be employed. In particular embodiments,leucine residues in a leucine zipper moiety are replaced by isoleucineresidues. Such peptides comprising isoleucine may be referred to asisoleucine zippers, but are encompassed by the term “leucine zippers” asemployed herein.

As described in example 7, a soluble Flag®-TRAIL polypeptide expressedin CV-1/EBNA cells spontaneously formed oligomers believed to be amixture of dimers and trimers. The cytotoxic effect of this solubleFlag®-TRAIL in the assay of example 8 was enhanced by including ananti-Flag® antibody, possibly because the antibody facilitatedcross-linking of TRAIL/receptor complexes. In one embodiment of theinvention, biological activity of TRAIL is enhanced by employing TRAILin conjunction with an antibody that is capable of cross-linking TRAIL.Cells that are to be killed may be contacted with both a soluble TRAILpolypeptide and such an antibody.

As one example, cancer or virally infected cells are contacted with ananti-Flag® antibody and a soluble Flag®-TRAIL polypeptide. Preferably,an antibody fragment lacking the Fc region is employed. Bivalent formsof the antibody may bind the Flag® moieties of two soluble Flag®-TRAILpolypeptides that are found in separate dimers or trimers. The antibodymay be mixed or incubated with a Flag®-TRAIL polypeptide prior toadministration in vivo. When an LZ-TRAIL protein is employed, anantibody directed against the leucine zipper peptide may be substitutedfor the anti-Flag® antibody, in the foregoing procedures.

Oligomerization is attributable to factors and mechanisms that include,but are not limited to, inter-chain disulfide bonds, and non-covalentinteractions such as hydrophobic interactions, as discussed above. Suchfactors and mechanisms influence the type of oligomers that are formed,which may include higher order oligomers, and may result in proteinpreparations comprising multiple species of oligomers (e.g., dimers,trimers, hexamers, 12-mers, and so on).

Provided herein are methods for manipulating oligomerization of TRAILand TRAIL-containing fusion proteins. The products of these methods alsoare provided.

One approach involves altering the number of cysteine residues in aTRAIL polypeptide or fusion protein. The number of cysteines may beincreased or decreased, depending upon whether a corresponding increaseor decrease in disulfide bonds is desired.

One may choose to inhibit or promote disulfide bond formation, dependingon the form of TRAIL that is desired for a particular purpose. Onereason for manipulating disulfide bond formation may be to obtain a morehomogeneous protein preparation, by controlling one mechanism ofoligomerization. The proportion of oligomers that are of a desiredspecies may be increased through such an approach. Another reason may beto enhance the proportion of a particularly advantageous form of TRAILin a protein preparation, such as an oligomeric form exhibiting enhancedbiological activity.

The amino acid sequence of a TRAIL protein or fusion protein may bealtered to increase or decrease the number of cysteine residues. Suchsequence alteration may be accomplished by conventional procedures, suchas mutagenesis techniques, as discussed above. One alternative forincreasing the number of cysteine residues involves addingcysteine-containing peptides, preferably fused to the N-terminus of aTRAIL polypeptide (or included in a fusion protein comprising TRAIL,such as an LZ-TRAIL fusion).

The cysteine residue at position 230 of SEQ ID NO:2 is located withinthe extracellular domain, which contains the receptor-binding region.One embodiment of the invention is directed to TRAIL polypeptides inwhich the Cys-230 residue is deleted or substituted. Formation ofdisulfide bonds involving the Cys-230 residue, including intramoleculardisulfides which would occur in the extracellular domain containing thereceptor-binding function of the protein, thus is avoided.

Oligomers may be treated with chemical cross-linking reagents. Reagentsthat stabilize the oligomers, without destroying a desired biologicalactivity, are chosen for use.

Expression Systems

The present invention provides recombinant expression vectors forexpression of TRAIL, and host cells transformed with the expressionvectors. Any suitable expression system may be employed. The vectorsinclude a DNA encoding a TRAIL polypeptide, operably linked to suitabletranscriptional or translational regulatory nucleotide sequences, suchas those derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are operably linked when theregulatory sequence functionally relates to the TRAIL DNA sequence.Thus, a promoter nucleotide sequence is operably linked to an TRAIL DNAsequence if the promoter nucleotide sequence controls the transcriptionof the TRAIL DNA sequence. An origin of replication that confers theability to replicate in the desired host cells, and a selection gene bywhich transformants are identified, are generally incorporated into theexpression vector.

In addition, a sequence encoding an appropriate signal peptide can beincorporated into expression vectors. A DNA sequence for a signalpeptide (secretory leader) may be fused in frame to the TRAIL sequenceso that the TRAIL is initially translated as a fusion protein comprisingthe signal peptide. A signal peptide that is functional in the intendedhost cells promotes extracellular secretion of the TRAIL polypeptide.The signal peptide is cleaved from the TRAIL polypeptide upon secretionof TRAIL from the cell.

Suitable host cells for expression of TRAIL polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-freetranslation systems could also be employed to produce TRAIL polypeptidesusing RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a TRAIL polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant TRAIL polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a TRAIL DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

TRAIL alternatively may be expressed in yeast host cells, preferablyfrom the Saccharomyces genus (e.g., S. cerevisiae). Other genera ofyeast, such as Pichia or Kluyveromyces, may also be employed. Yeastvectors will often contain an origin of replication sequence from a 2μyeast plasmid, an autonomously replicating sequence (ARS), a promoterregion, sequences for polyadenylation, sequences for transcriptiontermination, and a selectable marker gene. Suitable promoter sequencesfor yeast vectors include, among others, promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phospho-glucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657. Another alternative is the glucose-repressible ADH2 promoterdescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast andE. coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Amp^(r) gene and origin ofreplication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the TRAIL polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant TRAIL polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

CHO cells are preferred for use as host cells. One example of a suitableCHO cell line is the cell line designated DX-B11, which is deficient indihydrofolate reductase (DHFR), as described in Urlaub and Chasin (Proc.Natl. Acad. Sci USA 77:4216-4220, 1980), hereby incorporated byreference. DX-B11 cells may be transformed with expression vectors thatencode DHFR, which serves as a selectable marker (Kauffman et al., Meth.in Enzymology, 185:487-511, 1990). The use of DHFR as a selectablemarker, when cells are cultured in medium containing methotrexate, andfor amplifying a heterologous DNA inserted into the expression vector,are well known.

In other embodiments, the host cells are CHO cells that can be grown insuspension culture, and that are adapted to grow in media that does notcontain serum. The cells may be further adapted to grow in media lackinginsulin-like growth factor (IGF-1) and/or transferrin. The host cellsmay be adapted to grow in media that does not contain any exogenousgrowth factors that are animal proteins.

Such CHO cell lines may be generated by any suitable procedure. One suchprocedure is conducted generally as follows. DX-B11 cells are adapted togrowth in serum free medium by a gradual reduction of serumsupplementation in the media, and replacement of serum with low levelsof the growth factors transferrin and insulin-like growth factor(IGF-1), in an enriched cell growth media. Cells adapted to serum-freemedium then are weaned off transferrin and insulin-like growth factor-1.The resulting CHO cells maintain vigorous growth and high viability, aswell as a DHFR-deficient phenotype, in serum-free, essentiallyprotein-free, media.

Transformed host cells provided herein include, but are not limited to,host cells in which heterologous DNA, including a TRAIL-encodingsequence, is inserted into the cell's genomic DNA. Procedures thatresult in integration of expression vectors (or portions thereof) intohost cell DNA are well known. Conventional procedures may be employed toamplify, or increase the copy number of, heterologous DNA integratedinto the genomic DNA of transformed host cells.

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Expression vectors for use in mammalian host cells can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983), forexample. A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A high expression vector, PMLSV N1/N4, described by Cosman etal., Nature 312:768, 1984 has been deposited as ATCC 39890. Additionalmammalian expression vectors are described in EP-A-0367566, and in WO91/18982. As one alternative, the vector may be derived from aretrovirus. Additional suitable expression systems are described in theexamples below.

One preferred expression system employs Chinese hamster ovary (CHO)cells and an expression vector designated PG5.7. This expression vectoris described in U.S. patent application Ser. No. 08/586,509, filed Jan.11, 1996, and in PCT application publication no. WO 97/25420, which arehereby incorporated by reference. PG5.7 components include a fragment ofCHO cell genomic DNA, followed by a CMV-derived promoter, which isfollowed by a sequence encoding an adenovirus tripartite leader, whichin turn is followed by a sequence encoding dihydrofolate reductase(DHFR). These components were inserted into the plasmid vector pGEM1(Promega, Madison, Wis.). DNA encoding a TRAIL polypeptide (or fusionprotein containing TRAIL) may be inserted between the sequences encodingthe tripartite leader and DHFR. Methotrexate may be added to the culturemedium to increase expression levels, as is recognized in the field.

The fragment of CHO cell genomic DNA in vector PG5.7 enhances expressionof TRAIL. A phage lysate containing a fragment of genomic DNA isolatedfrom CHO cells was deposited with the American Type Culture Collectionon Jan. 4, 1996, and assigned accession number ATCC 97411. Vector PG5.7contains nucleotides 8671 through 14507 of the CHO genomic DNA insert instrain deposit ATCC 97411.

A further example of a suitable expression vector is similar to PG5.7,but comprises a multiple cloning site and an internal ribosome bindingsite (IRES), positioned between the adenovirus tripartite leader andDHFR-encoding sequences. The multiple cloning site comprises severalrestriction endonuclease recognition sites, at which heterologous DNA(e.g., TRAIL DNA) may be inserted into the vector. The IRES, a 575 bpnon-coding region derived from the encephalomyocarditis virus, allowscap-independent internal binding of the ribosome and initiation oftranslation. For discussion of the use of IRES sequences in expressionvectors, including the role such sequences play in allowing dicistronicmRNAs to be translated efficiently, see Kaufman R., Nucleic AcidsResearch 19:4485, 1991; Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295-300, 1993; and Ramesh et al., Nucleic Acids Research,24:2697-2700, 1996. In addition, the vector may comprise a truncated CHOgenomic DNA fragment, shorter than the fragment incorporated into PG5.7,yet still functional in enhancing expression of TRAIL (see WO 97/25420).

For expression of TRAIL, a type II protein lacking a native signalsequence, a heterologous signal sequence or leader functional inmammalian host cells may be added. Examples include the signal sequencefor interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, thesignal sequence for interleukin-2 receptor described in Cosman et al.,Nature 312:768 (1984); the interleukin-4 receptor signal peptidedescribed in EP 367,566; the type I interleukin-1 receptor signalpeptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846. Anotheroption is a leader derived from Ig-kappa, such as a leader comprisingthe amino acid sequenceMet-Gly-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-Gly-Ser-Thr-Gly(SEQ ID NO:25). Further alternatives are cytomegalovirus-derived leadersand signal peptides derived from a growth hormone, as described in moredetail below.

A preferred expression system employs a leader sequence derived fromcytomegalovirus (CMV). Example 7 illustrates the use of one such leader.In example 7, mammalian host cells were transformed with an expressionvector encoding the peptide Met Ala Arg Arg Leu Trp Ile Leu Ser Leu LeuAla Val Thr Leu Thr Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg ArgThr Ser Ser (SEQ ID NO:9) fused to the N-terminus of an octapeptidedesignated FLAG® (SEQ ID NO:7, described above), which in turn is fusedto the N-terminus of a soluble TRAIL polypeptide. Residues 1 through 29of SEQ ID NO:9 constitute a CMV-derived leader sequence, whereasresidues 30 through 32 are encoded by oligonucleotides employed inconstructing the expression vector described in example 7. In oneembodiment, DNA encoding a poly-His peptide (e.g., a peptide containingsix histidine residues) is positioned between the sequences encoding theCMV leader and the FLAG® peptide.

In another embodiment of the invention, the FLAG® peptide in tie fusionprotein described immediately above is replaced with a leucine zipperpeptide. Thus, one recombinant expression vector provided hereincomprises DNA encoding a fusion protein comprising a CMV leader, aleucine zipper peptide, and a soluble TRAIL polypeptide. One example ofsuch a fusion protein is depicted in FIG. 4 (SEQ ID NO: 13). The proteinof FIG. 4 comprises (from N- to C-terminus) a CMV leader (residues 1through 29 of SEQ ID NO:9); an optional tripeptide Thr-Ser-Ser encodedby oligonucleotides employed in vector construction (residues 30 through32 of SEQ ID NO:9); a leucine zipper (SEQ ID NO: 15); an optionaltripeptide Thr-Arg-Ser encoded by oligonucleotides employed in vectorconstruction, and amino acids 95 to 281 of the human TRAIL protein ofSEQ ID NO:2.

Expression systems that employ such CMV-derived leader peptides areuseful for expressing proteins other than TRAIL. Expression vectorscomprising a DNA sequence that encodes amino acids 1 through 29 of SEQID NO:9 are provided herein. In another embodiment, the vector comprisesa sequence that encodes amino acids 1 through 28 of SEQ ID NO:9. DNAencoding a desired heterologous protein is positioned downstream of, andin the same reading frame as, DNA encoding the leader. Additionalresidues (e.g., those encoded by linkers or primers) may be encoded byDNA positioned between the sequences encoding the leader and the desiredheterologous protein, as illustrated by the vector described in example7. As is understood in the pertinent field, the expression vectorscomprise promoters and any other desired regulatory sequences, operablylinked to the sequences encoding the leader and heterologous protein.

The leader peptide presented in SEQ ID NO:9 may be cleaved after thearginine residue at position 29 to yield the mature secreted form of aprotein fused thereto. Alternatively or additionally, cleavage may occurbetween amino acids 20 and 21, or between amino acids 28 and 29, of SEQID NO:9.

The skilled artisan will recognize that the position(s) at which thesignal peptide is cleaved may vary according to such factors as the typeof host cells employed, whether murine or human TRAIL is expressed bythe vector, and the like. Analysis by computer program reveals that theprimary cleavage site may be between residues 20 and 21 of SEQ ID NO:9.Cleavage between residues 22 and 23, and between residues 27 and 28, ispredicted to be possible, as well. To illustrate, expression andsecretion of a soluble murine TRAIL polypeptide resulted in cleavage ofa CMV-derived signal peptide at multiple positions. The three mostprominent species of secreted protein (in descending order) resultedfrom cleavage between amino acids 20 and 21 of SEQ ID NO:9, cleavagebetween amino acids 22 and 23, and cleavage between amino acids 27 and28.

In one particular expression system, in which the fusion protein of FIG.4 (SEQ ID NO:13) was expressed in CHO cells, the CMV leader was cleavedat two positions. Two forms of mature protein resulted, one comprisingamino acids 21 to 256, and the other comprising amino acids 29 to 256,of SEQ ID NO:13.

A signal peptide comprising amino acids 1 to 20 of the CMV leader of SEQID NO:9 is also provided herein. Such a signal peptide may yield a morehomogeneous preparation of mature protein, since certain of theabove-mentioned alternative signal peptidase cleavage sites are omittedfrom the leader.

A method for producing a heterologous recombinant protein involvesculturing mammalian host cells transformed with such an expressionvector under conditions that promote expression and secretion of theheterologous protein, and recovering the protein from the culturemedium. Expression systems employing CMV leaders may be used to produceany desired protein, examples of which include, but are not limited to,colony stimulating factors, interferons, interleukins, other cytokines,and cytokine receptors.

A particularly preferred signal peptide for expression of TRAILpolypeptides is a signal peptide derived from a growth hormone gene. Onesuch signal peptide or leader, which is derived from human growthhormone, comprises the following amino acid sequence: Met Ala Thr GlySer Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp Leu GlnGlu Gly Ser Ala (SEQ ID NO:19). An example of a DNA sequence thatencodes this growth hormone leader is as follows:

ATGGCTACAGGCTCCCGGACGTCCCTGTCCTGGCTTTTTGGCCTGCTCTGCCTGCCCTGGCTTCAAGAGGGCAGTGCA(SEQ ID NO:18).

One expression system employing such a signal peptide is describedbelow, in example 14. In alternative embodiments of the invention, CHOcells (described above) are employed as host cells, in place of theCV1-EBNA cells described in example 14. Preferred embodiments of thepresent invention are directed to expression vectors encoding a fusionprotein comprising (from N- to C-terminus) a growth hormone leader, theleucine zipper peptide of SEQ ID NO:15, and a soluble TRAIL polypeptide.In one embodiment, the TRAIL polypeptide is a soluble human TRAILpolypeptide comprising amino acids 95 to 281 of SEQ ID NO:2. Optionally,peptide linkers (which may be encoded by DNA segments resulting from thevector construction technique, for example) are positioned between thegrowth hormone leader and the leucine zipper, or between the leucinezipper and TRAIL. The leucine zipper moiety promotes oligomerization ofthe fusion proteins.

One example of such a fusion protein is depicted in FIG. 3. The fusionprotein comprises (from N- to C-terminus) a growth hormone-derivedleader sequence (SEQ ID NO:19), followed by a tripeptide encoded by anoligonucleotide employed in vector construction (Thr-Ser-Ser), a leucinezipper peptide (SEQ ID NO:15), a tripeptide encoded by anoligonucleotide employed in vector construction (Thr-Arg-Ser), and asoluble human TRAIL polypeptide (amino acids 95 to 281 of SEQ ID NO:2).A DNA sequence encoding the fusion protein, and the amino acid sequenceof the fusion protein, are presented in SEQ ID NO:10 and 11,respectively.

Purified TRAIL Protein

The present invention provides purified TRAIL proteins, which may beproduced by recombinant expression systems as described above orpurified from naturally occurring cells. The desired degree of puritymay depend on the intended use of the protein. A relatively high degreeof purity is desired when the protein is to be administered in vivo, forexample. Advantageously, TRAIL polypeptides are purified such that noprotein bands corresponding to other proteins are detectable bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto TRAIL protein may be detected by SDS-PAGE, due to differentialglycosylation, variations in post-translational processing, and thelike, as discussed above. A preparation of TRAIL protein is consideredto be purified as long as no bands corresponding to different(non-TRAIL) proteins are visualized. TRAIL most preferably is purifiedto substantial homogeneity, as indicated by a single protein band uponanalysis by SDS-PAGE. The protein band may be visualized by silverstaining, Coomassie blue staining, or (if the protein is radiolabeled)by autoradiography.

One process for producing the TRAIL protein comprises culturing a hostcell transformed with an expression vector comprising a DNA sequencethat encodes TRAIL under conditions such that TRAIL is expressed. TheTRAIL protein is then recovered from the culture (from the culturemedium or cell extracts). As the skilled artisan will recognize,procedures for purifying the recombinant TRAIL will vary according tosuch factors as the type of host cells employed and whether or not theTRAIL is secreted into the culture medium.

For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify TRAIL. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea purified TRAIL protein.

In one example of a procedure for producing and purifying TRAIL, ChineseHamster Ovary (CHO) cells are stably transformed with a recombinantexpression vector encoding soluble TRAIL. In one embodiment, the vectorencodes a fusion protein comprising a CMV-derived leader, a leucinezipper, and a soluble TRAIL polypeptide, as described in more detailelsewhere herein. The transformed cells are cultured to allow expressionand secretion of the soluble LZ-TRAIL protein into the culturesupernatant. The culture supernatant then is diluted 5-fold with 20 mMTris buffer, pH 8.5, and applied to a Q-Sepharose anion exchange column(Pharmacia LKB, Uppsala, Sweden) at a ratio of 1 ml supernatant per 0.3ml bead volume. The flow-through then is passed over a Fractogel®S-Sepharose cation exhange column (EM Separations, Gibbstown, N.J.) at aratio of 1/0.06 ml (v/v), washed with five column volumes of buffer, andeluted with a salt gradient of 0 to 1.0M NaCl in 20 mM Tris buffer, pH8.5. Fractions containing the LZ/TRAIL protein are pooled and dialyzedagainst Tris Buffered Saline (TBS).

Another example of a protein purification procedure, slightly modifiedfrom the procedure described immediately above, is as follows. Thisprocedure may be employed when a leader derived from growth hormone issubstituted for the CMV leader, for example. Chinese Hamster Ovary (CHO)cells are stably transformed with a recombinant expression vectorencoding GH leader-LZ-TRAIL, and cultured to allow expression andsecretion of the soluble LZ-TRAIL protein into the culture supernatant.The culture supernatant then is diluted 5-fold with 25 mM Tris buffer,pH 7.0, and applied to a Q-Sepharose anion exchange column (PharmaciaLKB, Uppsala, Sweden) at a ratio of 1 ml supernatant per 0.3 ml beadvolume. The flow-through was concentrated and buffer exchanged into 10mM Tris, pH 7.0, then passed over a Fractogel® S-Sepharose cationexhange column (EM Separations, Gibbstown, N.J.) at a ratio of 1/0.06 ml(v/v); washed with five column volumes of buffer; and eluted with a 0 to0.5M NaCl gradient in 10 mM Tris buffer, pH 7.0. Fractions containingthe LZ/TRAIL protein were concentrated and applied to an S200 sizingcolumn (Pharmacia) in 10 mM Tris, pH 7.0, 100 mM NaCl, 10% glycerol.

When bacterial host cells are employed, the recombinant protein producedin bacterial culture may be isolated by initial disruption of the hostcells, centrifugation, extraction from cell pellets if an insolublepolypeptide, or from the supernatant fluid if a soluble polypeptide,followed by one or more concentration, salting-out, ion exchange,affinity purification or size exclusion chromatography steps. Finally,RP-HPLC can be employed for final purification steps. Microbial cellscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

When transformed yeast host cells are employed, TRAIL preferably isexpressed as a secreted polypeptide. This simplifies purification.Secreted recombinant polypeptide from a yeast host cell fermentation canbe purified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

Alternatively, TRAIL polypeptides can be purified by immunoaffinitychromatography. An affinity column containing an antibody that bindsTRAIL may be prepared by conventional procedures and employed inpurifying TRAIL. Example 4 describes a procedure for generatingmonoclonal antibodies directed against TRAIL.

Expression of various forms of TRAIL described herein in particularexpression systems may yield protein preparations comprising multiplespecies of oligomers (e.g., dimers, trimers, hexamers, 12-mers, and soon). To illustrate, a mixture of oligomers, including hexamers andtrimers, may result from expression of the fusion protein of SEQ IDNO:11 in COS cells. In another illustrative scenario, involvingexpression of a fusion protein comprising a growth hormone leader, anisoleucine zipper and a spacer-deleted soluble TRAIL polypeptide,12-mers may be among the resulting oligomers. If a particular species ofoligomer is desired for a particular use, that species may be isolatedusing conventional procedures. An example of a suitable procedureemploys size exclusion chromatography.

A TRAIL protein (e.g., a fusion protein or oligomer) prepared using aparticular expression system may comprise inter- or intra-moleculardisulfide bonds that are disadvantageous for a particular use of theprotein. In such a case, the TRAIL protein may be treated with areducing agent in accordance with conventional techniques. An example ofa suitable procedures comprises treating the protein with 5-10 mM DTT(dithiothreitol) for 10 minutes at 37° C. Other suitable reducingagents, such as B-mercaptoethanol (preferably at a concentration of atleast 100 mM in the reaction solution), may be substituted for DTT andused in accordance with standard procedures.

To inhibit reoxidation and formation of new disulfide bonds, the proteinmay be stored in the presence of a reducing agent. One alternativeinvolves further treatment of the protein, after the reducing step, witha sulfhydryl-specific modifying agent. Examples of such agents areiodoacetamide or iodoacetic acid.

Treatment with a reducing agent may be conducted when refolding of aprotein into a different conformation is desired. Disulfide bonds,including intramolecular disulfide bonds, are reduced, and the reducingagent then removed to allow refolding of the protein. If promotingdisulfide bond formation is desired, oxygen can be bubbled through thereaction solution.

Properties and Uses of TRAIL

Programmed cell death (apoptosis) occurs during embryogenesis,metamorphosis, endocrine-dependent tissue atrophy, normal tissueturnover, and death of immune thymocytes. Regulation of programmed celldeath is vital for normal functioning of the immune system. Toillustrate, T cells that recognize self-antigens are destroyed throughthe apoptotic process during maturation of T-cells in the thymus,whereas other T cells are positively selected. The possibility that someT-cells recognizing certain self epitopes (e.g., inefficiently processedand presented antigenic determinants of a given self protein) escapethis elimination process and subsequently play a role in autoimmunediseases has been proposed (Gammon et al., Immunology Today 12:193,1991).

Insufficient apoptosis has been implicated in certain conditions, whileelevated levels of apoptotic cell death have been associated with otherdiseases. The desirability of identifying and using agents that regulateapoptosis in treating such disorders is recognized (Kromer, Advances inImmunology, 58:211, 1995; Groux et al., J. Exp. Med. 175:331, 1992;Sachs and Lotem, Blood 82:15, 1993).

Abnormal resistance of T cells toward undergoing apoptosis has beenlinked to lymphocytosis, lymphadenopathy, splenomegaly, accumulation ofself-reactive T cells, autoimmune disease, development of leukemia, anddevelopment of lymphoma (Kromer, supra; see especially pages 214-215).Conversely, excessive apoptosis of T cells has been suggested to play arole in lymphopenia, systemic immunodeficiency, and specificimmunodeficiency, with specific examples being virus-inducedimmunodeficient states associated with infectious mononucleosis andcytomegalovirus infection, and tumor-mediated immunosuppression (Kromer,supra; see especially page 214). Depletion of CD4⁺ T cells inHIV-infected individuals may be attributable to inappropriateactivation-induced cell death (AICD) by apoptosis (Groux et al., J. Exp.Med. 175:331, 1992).

As demonstrated in examples 5 and 8, TRAIL induces apoptosis of theacute T cell leukemia cell line designated Jurkat clone E6-1. TRAIL thusis a research reagent useful in studies of apoptosis, including theregulation of programmed cell death. Since Jurkat cells are a leukemiacell line arising from T cells, the TRAIL of the present invention findsuse in studies of the role TRAIL may play in apoptosis of othertransformed T cells, such as other malignant cell types arising from Tcells.

TRAIL binds Jurkat cells, as well as inducing apoptosis thereof. TRAILdid not cause death of freshly isolated murine thymocytes, or peripheralblood T cells (PBTs) freshly extracted from a healthy human donor. Anumber of uses flow from these properties of TRAIL.

TRAIL polypeptides may be used to purify leukemia cells, or any othercell type to which TRAIL binds. Leukemia cells may be isolated from apatient's blood, for example. In one embodiment, the cells are purifiedby affinity chromatography, using a chromatography matrix having TRAILbound thereto. The TRAIL attached to the chromatography matrix may be afull length protein, an TRAIL fragment comprising the extracellulardomain, an TRAIL-containing fusion protein, or other suitable TRAILpolypeptide described herein. In one embodiment, a soluble TRAIL/Fcfusion protein is bound to a Protein A or Protein G column throughinteraction of the Fc moiety with the Protein A or Protein G.Alternatively, TRAIL may be used in isolating leukemia cells by flowcytometry.

The thus-purified leukemia cells are expected to die following bindingof TRAIL, but the dead cells will still bear cell surface antigens, andmay be employed as immunogens in deriving anti-leukemia antibodies. Theleukemia cells, or a desired cell surface antigen isolated therefrom,find further use in vaccine development.

Since TRAIL binds and kills leukemia cells (the Jurkat cell line), TRAILalso may be useful in treating leukemia. A therapeutic method involvescontacting leukemia cells with an effective amount of TRAIL. In oneembodiment, a leukemia patient's blood is contacted ex vivo with anTRAIL polypeptide. The TRAIL may be immobilized on a suitable matrix.TRAIL binds the leukemia cells, thus removing them from the patient'sblood before the blood is returned into the patient.

Alternatively or additionally, bone marrow extracted from a leukemiapatient may be contacted with an amount of TRAIL effective in inducingdeath of leukemia cells in the bone marrow. Bone marrow may be aspiratedfrom the sternum or iliac crests, for example, and contacted with TRAILto purge leukemia cells. The thus-treated marrow is returned to thepatient.

TRAIL also binds to, and induces apoptosis of, lymphoma and melanomacells (see examples 5, 9, and 10). Thus, uses of TRAIL that areanalogous to those described above for leukemia cells are applicable tolymphoma and melanoma cells. TRAIL polypeptides may be employed intreating cancer, including, but not limited to, leukemia, lymphoma, andmelanoma. In one embodiment, the lymphoma is Burkitt's lymphoma. Table Iin example 9 shows that TRAIL had a cytotoxic effect on severalBurkitt's lymphoma cell lines. Epstein-Barr virus is an etiologic agentof Burkitt's lymphoma.

TRAIL polypeptides also find use in treating viral infections. Contactwith TRAIL caused death of cells infected with cytomegalovirus, but notof the same cell type when uninfected, as described in example 11. Theability of TRAIL to kill cells infected with other viruses can beconfirmed using the assay described in example 11. Such viruses include,but are not limited to, encephalomyocarditis virus, Newcastle diseasevirus, vesicular stomatitis virus, herpes simplex virus, adenovirus-2,bovine viral diarrhea virus, HIV, and Epstein-Barr virus.

An effective amount of TRAIL is administered to a mammal, including ahuman, afflicted with a viral infection. In one embodiment, TRAIL isemployed in conjunction with interferon to treat a viral infection. Inthe experiment described in example 11, pretreatment of CMV-infectedcells with γ-interferon enhanced the level of killing of the infectedcells that was mediated by TRAIL. TRAIL may be administered inconjunction with other agents that exert a cytotoxic effect on cancercells or virus-infected cells.

A wide variety of drugs have been employed in cancer treatment. Examplesinclude, but are not limited to, cisplatin, taxol, etoposide,Novantrone® (mitoxantrone), actinomycin D, camptothecin (or watersoluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycinC), dacarbazine (DTIC), and anti-neoplastic antibiotics such asdoxorubicin and daunomycin. Drugs employed in cancer therapy may have acytotoxic or cytostatic effect on cancer cells, or may reduceproliferation of the malignant cells. Cancer treatment may includeradiation therapy. In particular embodiments, TRAIL may beco-administered with other proteins in cancer therapy; one such proteinis γ-interferon.

Among the texts providing guidance for cancer therapy is Cancer,Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. J.B. Lippincott Co., Philadelphia, Pa. (1993). An appropriate therapeuticapproach is chosen according to the particular type of cancer, and otherfactors such as the general condition of the patient, as is recognizedin the pertinent field.

TRAIL may be added to a standard chemotherapy regimen, in treating acancer patient. For those combinations in which TRAIL and a secondanti-cancer agent exert a synergistic effect against cancer cells, thedosage of the second agent may be reduced, compared to the standarddosage of the second agent when administered alone. A method forincreasing the sensitivity of cancer cells to TRAIL comprisesco-administering TRAIL with an amount of a chemotherapeutic anti-cancerdrug that is effective in enhancing sensitivity of cancer cells toTRAIL.

Particular embodiments of the invention are directed toco-administration of TRAIL and methotrexate, etoposide, or mitoxantroneto a cancer patient, including but not limited to prostate cancerpatients. One such therapeutic method comprises administration of TRAILand mitoxantrone (Novantrone®; Immunex Corporation, Seattle, Wash.) to aprostate cancer patient. For descriptions of mitoxantrone or the usethereof in treating prostate cancer, see U.S. Pat. Nos. 4,197,249 and4,278,689; and Moore et al. (J. Clinical Oncology 12:689-694, 1994),which are hereby incorporated by reference. In an in vitro assay inwhich a prostate tumor cell line was contacted with variousconcentrations of LZ-TRAIL and Novantrone®, a synergistic effect wasseen, in that the combination of LZ-TRAIL and Novantrone® resulting inenhanced tumor cell death. A synergistic effect also was seen when TRAILand methotrexate were employed in the assay. LZ-TRAIL is a fusionprotein comprising a leucine zipper peptide and a soluble TRAILpolypeptide, as described in more detail above and in example 14.

Another embodiment of the invention is directed to contacting colorectalcancer cells (e.g., colon carcinoma cells) with TRAIL and camptothecene.Alternatives include contacting colorectal cancer cells with TRAIL inconjunction with adriamycin (doxorubicin) or mitomycin.

For in vivo use, derivatives of camptothecene that are more watersoluble would be advantageous. Examples of such water solublederivatives are the drugs7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin(CPT-11; irinotecan) and 9-dimethyl-aminomethyl-10-hydroxycamptothecin(topotecan). Camptothecene and the two above-described derivatives areDNA topoisomerase I inhibitors.

A method for treatment of colorectal cancer (e.g., colon carcinoma)comprises administering TRAIL in conjunction with a water solublederivative of camptothecene, such as irinotecan or topotecan. Forfurther description of the chemical structure of irinotecan andtopotecan, or the use thereof in treating cancer, including colorectalcancer, see Rougier et al. (J. Clinical Oncology, 15:251-260, January1997), Pommier (Seminars in Oncology Vol. 23, No. 1, Suppl. 3, pp 3-10,February 1996), Lavelle et al. (Seminars in Oncology Vol. 23, No. 1,Suppl. 3, pp 11-20, February 1996), Pitot et al. (J. Clinical Oncology,15:2910-2919, August 1997), Kunimoto et al. (Cancer Research,47:5944-5947, Nov. 15, 1987), and Jansen et al. (Int. J. of Cancer,70:335-40, Jan. 27, 1997), all hereby incorporated by reference.

Also provided herein are methods for treating melanoma by administeringTRAIL in conjunction with other therapeutic agent(s). In an in vitroassay, actinomycin D and cycloheximide were found to enhance thesensitivity of certain melanoma cell lines to TRAIL. For particularmelanoma cell lines that were resistant to TRAIL-mediated cytotoxicity,addition of the protein synthesis inhibitors actinomycin D orcycloheximide rendered the cells more sensitive to TRAIL-induced death.Thus, one method of the present invention comprises co-administeringTRAIL, together with actinomycin D or cycloheximide, to a melanomapatient.

Experimental evidence suggests a correlation between the intracellularconcentration of an apoptosis inhibitor designated FLIP, and sensitivityof certain tumor cells to TRAIL-mediated cell death. CertainTRAIL-resistant melanoma cell lines expressed relatively high levels ofFLIP, whereas lower levels (or undetectable levels) of FLIP wereexpressed in TRAIL-sensitive melanoma cell lines. Further, addition ofactinomycin D to certain TRAIL-resistant melanoma cell lines resulted ina decrease in the intracellular concentration of FLIP. For furtherdiscussion of FLIP proteins (FLICE inhibitory proteins) and theirinvolvement in caspase signaling cascades, see Thome et al., (Nature,386:517, 3 April 1997) and Irmler et al. (Nature 388:190, 1997), herebyincorporated by reference.

One approach toward increasing sensitivity of cancer cells (includingbut not limited to melanoma cells) to TRAIL is inhibiting expression ofFLIP in the target cancer cells. Antisense molecules that are derivedfrom a FLIP DNA sequence, and that will inhibit FLIP expression intarget cells, may be employed in such an approach.

As used herein, “co-administration” is not limited to simultaneousadministration. TRAIL may be administered along with other therapeuticagents, during the course of a treatment regimen. In one embodiment,administration of TRAIL and other therapeutic agents is sequential. Anappropriate time course may be chosen by the physician, according tosuch factors as the nature of a patient's illness, and the patient'scondition.

In preferred embodiments of the therapeutic methods described herein,soluble human TRAIL polypeptides, or oligomeric forms thereof, areadministered. Oligomers comprising leucine zipper-TRAIL fusion proteinsare especially preferred.

In another embodiment, TRAIL is used to kill virally infected cells incell preparations, tissues, or organs that are to be transplanted. Toillustrate, bone marrow may be contacted with TRAIL to kill virusinfected cells that may be present therein, before the bone marrow istransplanted into the recipient.

The TRAIL of the present invention may be used in developing treatmentsfor any disorder mediated (directly or indirectly) by defective orinsufficient amounts of TRAIL. A therapeutically effective amount ofpurified TRAIL protein is administered to a patient afflicted with sucha disorder. Alternatively, TRAIL DNA sequences may be employed indeveloping a gene therapy approach to treating such disorders.Disclosure herein of native TRAIL nucleotide sequences permits thedetection of defective TRAIL genes, and the replacement thereof withnormal TRAIL-encoding genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparision of the native TRAIL nucleotidesequence disclosed herein with that of a TRAIL gene derived from aperson suspected of harboring a defect in this gene.

The present invention provides pharmaceutical compositions comprisingpurified TRAIL and a physiologically acceptable carrier, diluent, orexcipient. Suitable carriers, diluents, and excipients are nontoxic torecipients at the dosages and concentrations employed. Such compositionsmay comprise buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) polypeptides, proteins, aminoacids, carbohydrates including glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione and other stabilizers and excipientscommonly employed in pharmaceutical compositions. Neutral bufferedsaline or saline mixed with conspecific serum albumin are among theappropriate diluents. The composition may be formulated as alyophilizate using appropriate excipient solutions (e.g. sucrose) asdiluents.

For therapeutic use, purified proteins of the present invention areadministered to a patient, preferably a human, for treatment in a mannerappropriate to the indication. Thus, for example, the pharmaceuticalcompositions can be administered locally, by intravenous injection,continuous infusion, sustained release from implants, or other suitabletechnique. Appropriate dosages and the frequency of administration willdepend, of course, on such factors as the nature and severity of theindication being treated, the desired response, the condition of thepatient and so forth.

The TRAIL protein employed in the pharmaceutical compositions preferablyis purified such that the TRAIL protein is substantially free of otherproteins of natural or endogenous origin, desirably containing less thanabout 1% by mass of protein contaminants residual of productionprocesses. Such compositions, however, can contain other proteins addedas stabilizers, carriers, excipients or co-therapeutics.

The TRAIL-encoding DNAs disclosed herein find use in the production ofTRAIL polypeptides, as discussed above. Fragments of the TRAILnucleotide sequences are also useful. In one embodiment, such fragmentscomprise at least about 17 consecutive nucleotides, more preferably atleast 30 consecutive nucleotides, of the human or murine TRAIL DNAdisclosed herein. DNA and RNA complements of said fragments are providedherein, along with both single-stranded and double-stranded forms of theTRAIL DNA of SEQ ID NOS:1, 3 and 5.

Among the uses of such TRAIL nucleic acid fragments are use as a probeor as primers in a polymerase chain reaction (PCR). As one example, aprobe corresponding to the extracellular domain of TRAIL may beemployed. The probes find use in detecting the presence of TRAIL nucleicacids in in vitro assays and in such procedures as Northern and Southernblots. Cell types expressing TRAIL can be identified as well. Suchprocedures are well known, and the skilled artisan can choose a probe ofsuitable length, depending on the particular intended application. ForPCR, 5′ and 3′ primers corresponding to the termini of a desired TRAILDNA sequence are employed to isolate and amplify that sequence, usingconventional techniques.

Other useful fragments of the TRAIL nucleic acids are antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target TRAIL MRNA (sense) orTRAIL DNA (antisense) sequences. Such a fragment generally comprises atleast about 14 nucleotides, preferably from about 14 to about 30nucleotides. The ability to create an antisense or a senseoligonucleotide, based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus maybe used to block expression of TRAIL proteins.

Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increases affinity of theoligonucleotide for a target nucleic acid sequence, suchas,poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oliginucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or other gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application WO 90/13641).Alternatively, other promotor sequences may be used to express theoligonucleotide.

Sense or antisense oligonucleotides may also be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antibodies Immunoreactive with TRAIL

The TRAIL proteins of the present invention, or immunogenic fragmentsthereof, may be employed in generating antibodies. The present inventionthus provides antibodies that specifically bind TRAIL, i.e., theantibodies bind to TRAIL via the antigen-binding sites of the antibody(as opposed to non-specific binding).

Polyclonal and monoclonal antibodies may be prepared by conventionaltechniques. See, for example, Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Kennet et al. (eds.), Plenum Press,New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land(eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1988). Production of monoclonal antibodies that are immunoreactive withTRAIL is further illustrated in example 4 below.

Antigen-binding fragments of such antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab, F(ab′),and F(ab′)₂ fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques, and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993).

Among the uses of the antibodies is use in assays to detect the presenceof TRAIL polypeptides, either in vitro or in vivo. The antibodies findfurther use in purifying TRAIL by affinity chromatography.

Those antibodies that additionally can block binding of TRAIL to targetcells may be used to inhibit a biological activity of TRAIL. Atherapeutic method involves in vivo administration of such an antibodyin an amount effective in inhibiting a TRAIL-mediated biologicalactivity. Disorders mediated or exacerbated by TRAIL, directly orindirectly, are thus treated. Monoclonal antibodies are generallypreferred for use in such therapeutic methods.

Antibodies directed against TRAIL may be useful for treating thromboticmicroangiopathies. One such disorder is thrombotic thrombocytopenicpurpura (TTP) (Kwaan, H. C., Semin. Hematol., 24:71, 1987; Thompson etal., Blood, 80:1890, 1992). Increasing TTP-associated mortality rateshave been reported by the U.S. Centers for Disease Control (Torok etal., Am. J. Hematol. 50:84, 1995).

Plasma from patients afflicted with TTP (including HIV⁺ and HIV⁻patients) induces apoptosis of human endothelial cells of dermalmicrovascular origin, but not large vessel origin (Laurence et al.,Blood, 87:3245, Apr. 15, 1996). Plasma of TTP patients thus is thoughtto contain one or more factors that directly or indirectly induceapoptosis. In the assay described in example 13 below, polyclonalantibodies raised against TRAIL inhibited TTP plasma-induced apoptosisof dermal microvascular endothelial cells. The data presented in example13 suggest that TRAIL is present in the serum of TTP patients, and mayplay a role in inducing apoptosis of microvascular endothelial cells.

Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS)(Moake, J. L., Lancet, 343:393, 1994; Melnyk et al., (Arch. Intern.Med., 155:2077, 1995; Thompson et al., supra). One embodiment of theinvention is directed to use of an anti-TRAIL antibody to treat thecondition that is often referred to as “adult HUS” (even though it canstrike children as well). A disorder known aschildhood/diarrhea-associated HUS differs in etiology from adult HUS.

Other conditions characterized by clotting of small blood vessels may betreated using anti-TRAIL antibodies. Such conditions include but are notlimited to the following. Cardiac problems seen in about 5-10% ofpediatric AIDS patients are believed to involve clotting of small bloodvessels. Breakdown of the microvasculature in the heart has beenreported in multiple sclerosis patients. As a further example, treatmentof systemic lupus erythematosus (SLE) is contemplated.

In one embodiment, a patient's blood or plasma is contacted with ananti-TRAIL antibody ex vivo. The antibody (preferably a monoclonalantibody) may be bound to a suitable chromatography matrix byconventional procedures. The patient's blood or plasma flows through achromatography column containing the antibody bound to the matrix,before being returned to the patient. The immobilized antibody bindsTRAIL, thus removing TRAIL protein from the patient's blood.

In an alternative embodiment, the antibodies are administered in vivo,in which case blocking antibodies are desirably employed. Suchantibodies may be identified using any suitable assay procedure, such asby testing antibodies for the ability to inhibit binding of TRAIL totarget cells. Alternatively, blocking antibodies may be identified inassays for the ability to inhibit a biological effect of the binding ofTRAIL to target cells. Example 12 illustrates one suitable method ofidentifying blocking antibodies, wherein antibodies are assayed for theability to inhibit TRAIL-mediated lysis of Jurkat cells.

The present invention thus provides a method for treating a thromboticmicroangiopathy, involving use of an effective amount of an antibodydirected against TRAIL. Antibodies of the present invention may beemployed in in vivo or ex vivo procedures, to inhibit TRAIL-mediateddamage to (e.g., apoptosis of) microvascular endothelial cells.

Anti-TRAIL antibodies may be employed in conjunction with other agentsuseful in treating a particular disorder. In an in vitro study reportedby Laurence et al. (Blood 87:3245, 1996), some reduction of TTPplasma-mediated apoptosis of microvascular endothelial cells wasachieved by using an anti-Fas blocking antibody, aurintricarboxylicacid, or normal plasma depleted of cryoprecipitate.

Thus, a patient may be treated with an agent that inhibitsFas-ligand-mediated apoptosis of endothelial cells, in combination withan agent that inhibits TRAIL-mediated apoptosis of endothelial cells. Inone embodiment, an anti-TRAIL blocking antibody and an anti-FAS blockingantibody are both administered to a patient afflicted with a disordercharacterized by thrombotic microangiopathy, such as TTP or HUS.Examples of blocking monoclonal antibodies directed against Fas antigen(CD95) are described in PCT application publication number WO 95/10540,hereby incorporated by reference.

Pharmaceutical compositions comprising an antibody that isimmunoreactive with TRAIL, and a suitable, diluent, excipient, orcarrier, are provided herein. Suitable components of such compositionsare as described above for the compositions containing TRAIL proteins.

The following examples are provided to illustrate particular embodimentsof the present invention, and are not to be construed as limiting thescope of the invention.

EXAMPLE 1

Isolation of a Human TRAIL DNA

DNA encoding a human TRAIL protein of the present invention was isolatedby the following procedure. A TBLASTN search of the dbEST data base atthe National Center for Biological Information (NCBI) was performed,using the query sequence LVVXXXGLYYVYXQVXF (SEQ ID NO:8). This sequenceis based upon the most conserved region of the TNF ligand family (Smithet al., Cell, 73:1349, 1993). An expressed sequence tag (EST) file,GenBank accession number Z36726, was identified using these searchparameters. The GenBank file indicated that this EST was obtained from ahuman heart atrium cDNA library.

Two 30-bp oligonucleotides based upon sequences from the 3′ and 5′ endsof this EST file were synthesized. The oligonucleotide from the 3′ endhad the sequence TGAAATCGAAAGTATGTTTGGGAATAGATG (complement ofnucleotides 636 to 665 of SEQ ID NO:1) and the 5′ oligonucleotide wasTGACGAAGAGAGTATGAACAGCCCCTGCTG (nucleotides 291 to 320 of SEQ ID NO:1).The oligonucleotides were 5′ end labeled with ³²P γ-ATP andpolynucleotide kinase. Two λgt10 cDNA libraries were screened byconventional methods with an equimolar mixture of these labeledoligonucleotides as probe. One library was a human heart 5′ stretch cDNAlibrary (Stratagene Cloning Systems, La Jolla, Calif.). The other was aperipheral blood lymphocyte (PBL) library prepared as follows: PBLs wereobtained from normal human volunteers and treated with 10 ng/ml of OKT3(an anti-CD3 antibody) and 10 ng/ml of human IL-2 for six days. The PBLcells were washed and stimulated with 500 ng/ml of ionomycin(Calbiochem) and 10 ng/ml PMA for 4 hours. Messenger RNA was isolatedfrom the stimulated PBL cells. cDNA synthesized on the mRNA template waspackaged into λgt10 phage vectors (Gigapak®, Stratagene Cloning Systems,La Jolla, Calif.).

Recombinant phages were plated onto E. coli strain C600-HFL and screenedusing standard plaque hybridization techniques. Nitrocellulose filterswere lifted from these plates in duplicate, and hybridized with the³²P-labeled oligonucleotides overnight at 67° C. in a solution of 60 mMTris pH 8.0, 2 mM EDTA, 5×Denhardt's Solution, 6×SSC, 1 mg/ml n-lauroylsarcosine, 0.5% NP40, and 4 μg/ml SS salmon sperm DNA. The filters werethen washed in 3×SSC at 67° C. for thirty minutes.

From the heart 5′ stretch cDNA library, one positive plaque was obtainedout of approximately one million plaques. This clone did not include the3′ end of the gene. Using the PBL library, approximately 50 positiveplaques were obtained out of 500,000 plaques. Fifteen of these firstround positive plaques were picked, and the inserts from the enrichedpools were amplified using oligonucleotide primers designed to amplifyphage inserts. The resulting products were resolved by 1.5% agarose gelelectrophoresis, blotted onto nitrocellulose, and analyzed by standardSouthern blot technique using the ³²P-labeled 30-mer oligonucleotides asprobes. The two plaque picks that produced the largest bands by Southernanalysis were purified by secondary screening, and isolated phageplaques were obtained using the same procedures described above.

DNA from the isolated phages was prepared by the plate lysis method, andthe cDNA inserts were excised with EcoRI, purified by electrophoresisusing 1.5% agarose in Tris-Borate-EDTA buffer, and ligated into thepBluescript® SK(+) plasmid. These inserts were then sequenced byconventional methods, and the resulting sequences were aligned.

The nucleotide sequence of a human TRAIL DNA is presented in SEQ ID NO:1and the amino acid sequence encoded thereby is presented in SEQ ID NO:2.This human TRAIL protein comprises an N-terminal cytoplasmic domain(amino acids 1-18), a transmembrane region (amino acids 19-38), and anextracellular domain (amino acids 39-281). The calculated molecularweight of this protein is 32,508 daltons.

E. coli strain DH10B cells transformed with a recombinant vectorcontaining this TRAIL DNA were deposited with the American Type CultureCollection on Jun. 14, 1995, and assigned accession no. 69849. Thedeposit was made under the terms of the Budapest Treaty. The recombinantvector in the deposited strain is the expression vector pDC409(described in example 5). The vector was digested with SalI and NotI,and human TRAIL DNA that includes the entire coding region shown in SEQID NO:1 was ligated into the digested vector.

EXAMPLE 2

Isolation of DNA Encoding a Truncated TRAIL

DNA encoding a second human TRAIL protein was isolated as follows. Thistruncated TRAIL does not exhibit the ability to induce apoptosis ofJurkat cells.

PCR analysis, using the 30-mers described in example 1 as the 5′ and 3′primers, indicated that 3 out of 14 of the first round plaque picks inexample 1 contained shorter forms of the TRAIL DNA. One of the shortenedforms of the gene was isolated, ligated into the pBluescript® SK(+)cloning vector (Stratagene Cloning Systems, La Jolla, Calif.) andsequenced.

The nucleotide sequence of this DNA is presented in SEQ ID NO:3. Theamino acid sequence encoded thereby is presented in SEQ ID NO:4. Theencoded protein comprises an N-terminal cytoplasmic domain (amino acids1-18), a transmembrane region (amino acids 19-38), and an extracellulardomain (amino acids 39-101).

The DNA of SEQ ID NO:3 lacks nucleotides 359 through 506 of the DNA ofSEQ ID NO:1, and is thus designated the human TRAIL deletion variant(huTRAILdv) clone. The deletion causes a shift in the reading frame,which results in an in-frame stop codon after amino acid 101 of SEQ IDNO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Aminoacids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through90 of SEQ ID NO:4. However, due to the deletion, the C-terminal portionof the huTRAILdv protein (amino acids 91 through 101 of SEQ ID NO:4)differs from the residues in the corresponding positions in SEQ ID NO:2.

The huTRAILdv protein lacks the above-described conserved regions foundat the C-terminus of members of the TNF family of proteins. Theinability of this huTRAILdv protein to cause apoptotic death of Jurkatcells further confirms the importance of these conserved regions forbiological activity.

EXAMPLE 3

DNA Encoding a Murine TRAIL

DNA encoding a murine TRAIL was isolated by the following procedure. AcDNA library comprising cDNA derived from the mouse T cell line 7B9 inthe vector λZAP was prepared as described in Mosley et al. (Cell 59:335,1989). DNA from the library was transferred onto nitrocellulose filtersby conventional techniques.

Human TRAIL DNA probes were used to identify hybridizing mouse cDNAs onthe filters. Two separate probes were used, in two rounds of screening.PCR reaction products about 400 bp in length, isolated and amplifiedusing the human TRAIL DNA as template, were employed as the probe in thefirst round of screening. These PCR products consisted of a fragment ofthe human TRAIL coding region. The probe used in the second round ofscreening consisted of the entire coding region of the human TRAIL DNAof SEQ ID NO:1. A random primed DNA labeling kit (Stratagene, La Jolla,Calif.) was used to radiolabel the probes.

Hybridization was conducted at 37° C. in 50% formamide, followed bywashing with 1×SSC, 0.1% SDS at 50° C. A mouse cDNA that was positive inboth rounds of screening was isolated.

The nucleotide sequence of this DNA is presented in SEQ ID NO:5 and theamino acid sequence encoded thereby is presented in SEQ ID NO:6. Theencoded protein comprises an N-terminal cytoplasmic domain (amino acids1-17), a transmembrane region (amino acids 18-38), and an extracellulardomain (amino acids 39-291). This mouse TRAIL is 64% identical to thehuman TRAIL of SEQ ID NO:2, at the amino acid level. The coding regionof the mouse TRAIL nucleotide sequence is 75% identical to the codingregion of the human nucleotide sequence of SEQ ID NO:1.

EXAMPLE 4

Antibodies that Bind TRAIL

This example illustrates the preparation of monoclonal antibodies thatspecifically bind TRAIL. Suitable immunogens that may be employed ingenerating such antibodies include, but are not limited to, purifiedTRAIL protein or an immunogenic fragment thereof (e.g., theextracellular domain), fusion proteins containing TRAIL polypeptides(e.g., soluble TRAIL/Fc fusion proteins), and cells expressingrecombinant TRAIL on the cell surface.

Known techniques for producing monoclonal antibodies include thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withTRAIL as an immunogen emulsified in complete Freund's adjuvant, andinjected in amounts ranging from 10-100 μg subcutaneously orintraperitoneally. Ten to twelve days later, the immunized animals areboosted with additional TRAIL emulsified in incomplete Freund'sadjuvant. Mice are periodically boosted thereafter on a weekly tobi-weekly immunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot blotassay or ELISA (Enzyme-Linked Immuno-sorbent Assay) for TRAILantibodies.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of TRAIL in saline. Three tofour days later, the animals are sacrificed, spleen cells harvested, andspleen cells are fused to a murine myeloma cell line such as NS1 or,preferably, P3x63Ag 8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified TRAIL by adaptations of the techniques disclosed in Engvall etal. (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-TRAILmonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can be used, as can affinity chromatography based upon bindingto TRAIL.

EXAMPLE 5

DNA Laddering Apoptosis Assay

Human TRAIL was expressed and tested for the ability to induceapoptosis. Oligonucleotides were synthesized that corresponded to the 3′and 5′ ends of the coding region of the human TRAIL gene, with SalI andNotI restriction sites incorporated at the ends of the oligonucleotides.The coding region of the human TRAIL gene was amplified by standard PCRtechniques, using these oligonucleotides as primers. The PCR reactionproducts were digested with the restriction endonucleases SalI and NotI,then inserted into SalI/NotI-digested vector pDC409. pDC409 is anexpression vector for use in mammalian cells, but is also replicable inE. coli cells.

pDC409 is derived from an expression vector designated pDC406 (describedin McMahan et al., EMBO J. 10:2821, 1991, and in PCT application WO91/18982, hereby incorporated by reference). pDC406 contains origins ofreplication derived from SV40, Epstein-Barr virus and pBR322 and is aderivative of HAV-EO described by Dower et al., J. Immunol. 142:4314(1989). pDC406 differs from HAV-EO by the deletion of an intron presentin the adenovirus 2 tripartite leader sequence in HAV-EO. DNA insertedinto a multiple cloning site (containing a number of restrictionendonuclease cleavage sites) is transcribed and translated usingregulatory elements derived from HIV and adenovirus. The vector alsocontains a gene that confers ampicillin resistance.

pDC409 differs from pDC406 in that a Bgl II site outside the mcs hasbeen deleted so that the mcs Bgl II site is unique. Two Pme 1 sites andone Srf 1 site have been added to the mcs, and three stop codons (TAG)have been positioned downstream of the mcs to function in all threereading frames. A T7 primer/promoter has been added to aid in the DNAsequencing process.

The monkey kidney cell line CV-1/EBNA-1 (ATCC CRL 10478) was derived bytransfection of the CV-1 cell line (ATCC CCL 70) with a gene encodingEpstein-Barr virus nuclear antigen-1 (EBNA-1) that constitutivelyexpresses EBNA-1 driven from the human CMV intermediate-earlyenhancer/promoter, as described by McMahan et al., supra. The EBNA-1gene allows for episomal replication of expression vectors, such aspDC409, that contain the EBV origin of replication.

CV1/EBNA cells grown in Falcon T175 flasks were transfected with 15 μgof either “empty” pDC409 or pDC409 containing the human TRAIL codingregion. The transformed cells were cultured for three days at 37° C. and10% CO₂. The cells then were washed with PBS, incubated for 20 minutesat 37° C. in 50 mM EDTA, scraped off of the flask with a cells scraper,and washed once in PBS. Next, the cells were fixed in 1%paraformaldehyde PBS for 10 minutes at 4° C., and washed 3× in PBS.

Jurkat cells were used as the target cells in this assay, to determinewhether the TRAIL-expressing cells could induce apoptosis thereof. TheJurkat cell line, clone E6-1, is a human acute T cell leukemia cell lineavailable from the American Type Culture Collection under accession no.ATCC TIB 152, and described in Weiss et al. (J. Immunol. 133:123-128,1984). The Jurkat cells were cultured in RPMI media supplemented with10% fetal bovine serum and 10 μg/ml streptomycin and penicillin to adensity of 200,000 to 500,000 cells per ml. Four million of these cellsper well were co-cultured in a 6 well plate with 2.5 mls of media withvarious combinations of fixed cells, supernatants from cells transfectedwith Fas ligand, and various antibodies, as indicated below.

After four hours the cells were washed once in PBS and pelleted at 1200RPM for 5 minutes in a desktop centrifuge. The pellets were resuspendedand incubated for ten minutes at 4° C. in 500 μl of buffer consisting of10 mM Tris-HCl, 10 mM EDTA, pH 7.5, and 0.2% Triton X-100, which lysesthe cells but leaves the nuclei intact. The lysate was then spun at 4°C. for ten minutes in a micro-centrifuge at 14,000 RPM. The supernatantswere removed and extracted three times with 1 ml of 25:24:1phenol-chloroform-isoamyl alcohol, followed by precipitation with NaOACand ethanol in the presence of 1 μg of glycogen carrier (Sigma).

The resulting pellets were resuspended in 10 mM Tris-HCl, 10 mM EDTA, pH7.5, and incubated with 10 μg/ml RNase A at 37° C. for 20 minutes. TheDNA solutions were then resolved by 1.5% agarose gel electrophoresis inTris-Borate EDTA buffer, stained with ethidium bromide and photographedwhile trans-illuminated with UV light.

The results were as follows. Fixed CV1/EBNA cells transfected witheither pDC409 or pDC409-TRAIL produced no detectable DNA laddering.pDC409-TRAIL fixed cells co-cultured with Jurkat cells produced DNAladdering, but pDC409 fixed cells co-cultured with Jurkat cells did not.

DNA laddering was also seen when Jurkat cells were co-cultured withconcentrated supernatants from COS cells transfected with DNA encodinghuman Fas ligand in pDC409. The supernatants are believed to containsoluble Fas ligand that is proteolytically released from the cellsurface. The Fas ligand-induced DNA laddering could be blocked by adding10 μg/ml of a soluble blocking monoclonal antibody directed against Fas.This same antibody could not inhibit laddering of Jurkat DNA by thepDC409-TRAIL cells, which indicates that TRAIL does not induce apoptosisthrough Fas.

In the same assay procedure, fixed CV1/EBNA cells transfected withpDC409-TRAIL induced DNA laddering in U937 cells. U937 (ATCC CRL 1593)is a human histiocytic lymphoma cell line. The ratio of effector totarget cells was 1:4 (the same as in the assay on Jurkat target cells).

The fragmentation of cellular DNA into a pattern known as DNA ladderingis a hallmark of apoptosis. In the foregoing assay, TRAIL inducedapoptosis of a leukemia cell line and a lymphoma cell line.

EXAMPLE 6

Northern Blot Analysis

Expression of TRAIL in a number of different tissue types was analysedin a conventional northern blot procedure. Northern blots containingpoly A³⁰ RNA from a variety of adult human tissues (multiple tissuenorthern blots I and II) were obtained from Clonetech (Palo Alto,Calif.). Other blots were prepared by resolving RNA samples on a 1.1%agarose-formaldehyde gel, blotting onto Hybond-N as recommended by themanufacturer (Amersham Corporation), and staining with methylene blue tomonitor RNA concentrations. The blots were probed with an antisense RNAriboprobe corresponding to the entire coding region of human TRAIL.

Human TRAIL mRNA was detected in peripheral blood lymphocytes, colon,small intestine, ovary, prostate, thymus, spleen, placenta, lung,kidney, heart, pancreas, and skeletal muscle. TRAIL transcripts werefound to be abundant in the large cell anaplastic lymphoma cell lineKarpas 299 (Fischer et al., Blood, 72:234, 1988) and in tonsilar Tcells. TRAIL message was present to a lesser degree in the Burkittlymphoma cell line designated Raji.

TRAIL mRNA was not detected in testis, brain, or liver, or in several Tcell lines. Little or no TRAIL transcripts were detected in freshlyisolated peripheral blood T cells (PBT), either unstimulated orstimulated with PMA and calcium ionophore for 20 hours.

EXAMPLE 7

Production of a Soluble TRAIL Polypeptide

A soluble human TRAIL polypeptide comprising amino acids 95 to 281 ofSEQ ID NO:2 was prepared as follows. This polypeptide is a fragment ofthe extracellular domain, lacking the spacer region discussed above.

An expression vector encoding soluble human TRAIL was constructed byfusing in-frame DNA encoding the following amino acid sequences (listedfrom N- to C-terminus): a leader sequence derived from humancytomegalovirus (CMV), a synthetic epitope designated Flag®, and aminoacids 95-281 of human TRAIL. The Flag® octapeptide (SEQ ID NO:7)facilitates purification of proteins fused thereto, as described aboveand in Hopp et al. (Biotechnology 6:1204-1210, 1988).

The TRAIL-encoding DNA fragment was isolated and amplified by polymerasechain reaction (PCR), using oligonucleotide primers that defined thetermini of a DNA fragment encoding amino acids 95-281 of SEQ ID NO:2.The 3′ primer was a 31-mer that additionally added a NotI sitedownstream of the TRAIL-encoding sequence. The 5′ primer added an SpeIsite and a Flag® epitope encoding sequence upstream of theTRAIL-encoding sequence. PCR was conducted by conventional procedures,using the above-described human TRAIL cDNA as the template.

The reaction products were digested with SpeI and NotI, and insertedinto the expression vector pDC409 (described in example 5), which hadbeen cleaved with SalI and NotI. Annealed oligonucleotides that form aSalI-SpeI fragment encoding a CMV open reading frame leader were alsoligated into the vector. The amino acid sequence of the CMV-derivedleader is presented as SEQ ID NO:9. Amino acids 1 to 29 of SEQ ID NO:9are encoded by CMV DNA, whereas amino acids 30 to 32 are encoded byoligonucleotides employed in constructing the vector. E. coli cells weretransfected with the ligation mixture, and the desired recombinantexpression vector was isolated therefrom.

CV1-EBNA cells (ATCC CRL 10478; described in example 5) were transfectedwith the recombinant vector, which is designated pDC409-Flag-shTRAIL,and cultured to allow expression and secretion of the solubleFlag®-TRAIL polypeptide. Culture supernatants were harvested 3 daysafter transfection and applied to a column containing an anti-Flag®antibody designated M2 immobilized on a solid support. The column thenwas washed with PBS. The monoclonal antibody M2 is described in Hopp etal., supra, and available from Kodak Scientific Imaging Systems, NewHaven, Conn. 800 μl fractions were eluted from the column with 50 mMcitrate, and immediately neutralized in 0.45 ml 1 M Tris (pH 8).Fractions were adjusted to 10% glycerol and stored at −20° C. untilneeded.

This soluble recombinant Flag®/human TRAIL expressed in CV1/EBNA cellshas an apparent molecular weight of 28 kD when analyzed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). The Flag® moietycontributes an estimated 880 daltons to the total molecular weight. Gelfiltration analysis of purified soluble Flag®/TRAIL suggests that themolecule is multimeric in solution with a size of ˜80 kD. While notwishing to be bound by theory, the gel filtration analysis suggests thatthe soluble recombinant Flag®/human TRAIL naturally formed a combinationof dimers and trimers, with trimers predominating.

An expression vector designated pDC409-Flag-smTRAIL, which encodes a CMVleader-Flag®-soluble murine TRAIL protein, was constructed by analogousprocedures. A DNA fragment encoding a soluble murine TRAIL polypeptidewas isolated and amplified by PCR. Oligonucleotides that defined thetermini of DNA encoding amino acids 99 to 291 of the murine TRAILsequence of SEQ ID NO:6 were employed as the 5′ and 3′ primers in thePCR.

EXAMPLE 8

Lysis of Leukemia Cells by Soluble TRAIL

In example 5, cells expressing human TRAIL induced apoptosis of Jurkatcells (a leukemia cell line). In the following study, a soluble humanTRAIL polypeptide killed Jurkat cells.

Jurkat cells were cultured to a density of 200,000 to 500,000 cells perml in RPMI medium supplemented with 10% fetal bovine serum, 100 μg/mlstreptomycin, and 100 μg/ml penicillin. The cells (in 96-well plates at50,000 cells per well in a volume of 100 μl) were incubated for twentyhours with the reagents indicated in FIG. 1. “TRAIL supe.” refers toconditioned supernatant (10 μl per well) from CV1/EBNA cells transfectedwith pDC409-Flag-shTRAIL (see example 7). “Control supe.” refers tosupernatant from CV1/EBNA cells transfected with empty vector. Whereindicated, immobilized anti-Flag® antibody M2 (“Imm. M2”) was added at aconcentration of 10 μg/ml in a volume of 100 μl per well and allowed toadhere either overnight at 4° C. or for 2 hours at 37° C., after whichwells were aspirated and washed twice with PBS to remove unboundantibody. Jurkat cells treated with Fas ligand or M3, a blockingmonoclonal antibody directed against Fas, (Alderson et al., J. Exp. Med.181:71, 1995; and PCT application WO 95/10540) were included in theassay as indicated.

Metabolic activity of the thus-treated cells was assayed by metabolicconversion of alamar Blue dye, in the following procedure. Alamar Blueconversion was measured by adding 10 μl of alamar Blue dye (BiosourceInternational, Camarillo, Calif.) per well, and subtracting the opticaldensity (OD) at 550-600 nm at the time the dye was added from the OD550-600 nm after four hours. No conversion of dye is plotted as 0percent viability, and the level of dye conversion in the absence ofTRAIL is plotted as 100 percent viability. Percent viability wascalculated by multiplying the ratio of staining of experimental versuscontrol cultures by 100.

The results are presented in FIG. 1. Error bars represent the standarddeviation of measurements from four independent wells, and the valuesare the average of these measurements.

The TRAIL-containing supernatant caused a significant reduction inviability of Jurkat cells. A greater reduction of cell viabilityresulted from contact with a combination of TRAIL-containing supernatantand immobilized anti-Flag® antibody M2. One possible explanation is thatM2 facilitates cross-linking of the Flag®/TRAIL-receptor complexes,thereby increasing the strength of signaling.

Fas ligand demonstrated the ability to kill Jurkat cells. The anti-Fasantibody M3 inhibited the activity of Fas ligand, but not the activityof TRAIL.

In order to confirm that the changes in dye conversion in the alamarBlue assay were due to cell death, the decrease in cell viabilityinduced by TRAIL was confirmed by staining the cells with trypan blue.

EXAMPLE 9

Lysis of Leukemia and Lymphoma Cells

In examples 5 and 8, TRAIL induced apoptosis of a leukemia cell line(Jurkat) and a lymphoma cell line (U937). The following study furtherdemonstrates the ability of TRAIL to kill leukemia and lymphoma cells.

The human cell lines indicated in Table I were cultured to a density of200,000 to 500,000 cells per ml in RPMI medium supplemented with 10%fetal bovine serum, 100 μg/ml streptomycin, and 100 μg/ml penicillin.The cells (in 96-well plates at 50,000 cells per well in a volume of 100μl) were incubated for twenty hours with conditioned supernatants (10 μlper well) from pDC409-Flag-shTRAIL transfected CV1/EBNA cells.

Metabolic activity was assayed by conversion of alamar Blue dye, in theassay procedure described in example 8. The results are presented inTable I.

In order to confirm that the changes in dye conversion in the alamarBlue assay were due to cell death, the decrease in cell viabilityinduced by TRAIL was confirmed by staining the cells with trypan blue.Crystal violet staining, performed as described by Flick and Gifford (J.Immunol. Methods 68:167-175, 1984), also confirmed the results seen inthe alamar Blue assay. The apoptotic nature of the cell death wasconfirmed by trypan blue staining and visualization of apoptoticfragmentation by microscopy.

As shown in Table I, many cancer cell lines were sensitive to TRAILmediated killing. The susceptibility of additional cell types to TRAILmediated apoptosis can be determined using the assay proceduresdescribed in this examples section.

TRAIL exhibited no significant cytotoxic effect on the cell lines THP-1K562, Karpas 299, and MP-1. K299, also known as Karpas 299, (DSM-ACC31)was established from peripheral blood of a male diagnosed with highgrade large cell anaplastic lymphoma (Fischer et al., Blood, 72:234,1988). MP-1 is a spontaneously derived EBV-transformed B cell line(Goodwin et al., Cell 73:447, 1993). While not wishing to be bound bytheory, it is possible that these four cell lines do not express areceptor for TRAIL, or are characterized by upregulation of a gene thatinhibits apoptosis.

TABLE 1 Effect of soluble TRAIL on cell line viability Cell LineDescription Percent Viability^(a) Bjab Burkitt lymphoma  0.5 ± 3.8 RamosBurkitt lymphoma 12.1 ± 2.1 U937 histiocytic lymphoma 25.2 ± 8.2 HL60promyelocytic leukemia 59.5 ± 3.2 Raji Burkitt lymphoma 64.9 ± 4.5 DaudiBurkitt lymphoma 70.2 ± 4.2 THP-1 monocytic cell line 92.3 ± 6.8 K562chronic myelogenous leukemia 97.1 ± 4.8 K 299 large cell anaplasticlymphoma 99.0 ± 4.3 MP-1 spontaneous B cell line 104.9 ± 11.7^(a)Results are means ± SEMs of 4 wells for each data point

EXAMPLE 10

Cross-Species Activity of TRAIL

Interspecies cross-reactivity of human and murine TRAIL was tested asfollows. Murine and human TRAIL were incubated with the human melanomacell line A375 (ATCC CRL 1619). Since this is an adherent cell line, acrystal violet assay, rather than alamar Blue, was used to determinecell viability. A375 cells were cultured in DMEM supplemented with 10%fetal bovine serum, 100 μg/ml streptomycin, and 100 μg/ml penicillin.The cells (in 96-well plates at 10,000 cells per well in a volume of 100μl) were incubated for 72 hours with the soluble murine TRAIL describedin example 7. Crystal violet staining was performed as described by(Flick and Gifford (J. Immunol. Methods 68:167-175, 1984). The resultsdemonstrated that both human and murine TRAIL are active on these humancells, in that murine and human TRAIL killed A375 cells.

The ability of human TRAIL to act on murine cells was tested, using theimmortalized murine fibroblast cell line L929. Incubation of L929 cellswith either human or murine TRAIL resulted in a decrease in crystalviolet staining, thus demonstrating that human and murine TRAIL areactive on (induced apoptosis of) murine cells. In addition to crystalviolet, cell death was confirmed by trypan-blue staining.

EXAMPLE 11

Lysis of CMV-Infected Cells

The following experiment demonstrates that the soluble Flag®-human TRAILprotein prepared in example 7 has a cytotoxic effect on virally infectedcells.

Normal human gingival fibroblasts were grown to confluency on 24 wellplates in 10% CO₂ and DMEM medium supplemented with 10% fetal bovineserum, 100 μg/ml streptomycin, and 100 μg/ml penicillin. Samples of thefibroblasts were treated as indicated in FIG. 2. Concentrations ofcytokines were 10 ng/ml for γ-interferon and 30 ng/ml of solubleFlag®-human TRAIL. All samples receiving TRAIL also received a two-foldexcess by weight of anti-Flag® antibody M2 (described above), whichenhances TRAIL activity (presumably by crosslinking).

Pretreatment of cells with the indicated cytokines was for 20 hours. Toinfect cells with cytomegalovirus (CMV), culture media were aspiratedand the cells were infected with CMV in DMEM with an approximate MOI(multiplicity of infection) of 5. After two hours the virus containingmedia was replaced with DMEM and cytokines added as indicated. After 24hours the cells were stained with crystal violet dye as described (Flickand Gifford, 1984, supra). Stained cells were washed twice with water,disrupted in 200 μl of 2% sodium deoxycholate, diluted 5 fold in water,and the OD taken at 570 nm. Percent maximal staining was calculated bynormalizing ODs to the sample that showed the greatest staining. Similarresults were obtained from several independent experiments.

The results presented in FIG. 2 demonstrate that TRAIL specificallykilled CMV infected fibroblasts. This cell death was enhanced bypretreatment of the cells with γ-interferon. No significant death ofnon-virally infected fibroblasts resulted from contact with TRAIL.

EXAMPLE 12

Assay to Identify Blocking Antibodies

Blocking antibodies directed against TRAIL may be identified by testingantibodies for the ability to inhibit a particular biological activityof TRAIL. In the following assay, a monoclonal antibody was tested forthe ability to inhibit TRAIL-mediated apoptosis of Jurkat cells. TheJurkat cell line is described in example 5.

A hybridoma cell line producing a monoclonal antibody raised against aFlag®/soluble human TRAIL fusion protein was employed in the assay.Supernatants from the hybridoma cultures were incubated with 20 ng/mlFlag®/soluble human TRAIL crosslinked with 40 ng/ml anti-Flag®monoclonal antibody M2, in RPMI complete media in a 96 well microtiterplate. An equivalent amount of fresh hybridoma culture medium was addedto control cultures. The Flag®/soluble human TRAIL fusion protein andthe monoclonal antibody designated M2 are described in example 7.

The hybridoma supernatant was employed at a 1:50 (v/v) dilution(starting concentration), and at two fold serial dilutions thereof.After incubation at 37° C., 10% CO₂, for 30 minutes, 50,000 Jurkat cellswere added per well, and incubation was continued for 20 hours.

Cell viability was then assessed measuring metabolic conversion ofalamar blue dye. An alamar blue conversion assay procedure is describedin example 8. The monoclonal antibody was found to inhibit the apoptosisof Jurkat cells induced by Flag®/soluble human TRAIL.

EXAMPLE 13

TRAIL Blocking Study

Human microvascular endothelial cells of dermal origin were treated for16-18 hours with plasma from patients with thrombotic thrombocytopenicpurpura (TTP) or with control plasma, either alone or in the presence ofanti-TRAIL polyclonal antiserum. A 1:2000 dilution of the antiserum wasemployed. The plasma was from two TTP patients, designated #1 and #2below, The cells employed in the assays were MVEC-1 (HMVEC 2753,purchased from Clonetics, San Diego, Calif.) and MVEC-2 (DHMVEC 30282,purchased from Cell Systems, Kirkland, Wash.). Cultures of these cellscan be maintained as described in Laurence et al. (Blood, 87:3245,1996).

The results were as follows. The data shown are from DNA histograms ofcells stained with propidium iodide, and “A₀ peak” represents theapoptotic peak (see Oyaizu et al., Blood, 82:3392, 1993; Nicoletti etal., J. Immunol. Methods, 139:271, 1991; and Laurence et al., Blood,75:696, 1990).

Microvascular EC Plasma (1%) Antibody % A₀ peak Experiment 1 DermalMVEC-1 control — 0 Dermal MVEC-1 TTP (#1) — 19.5 Dermal MVEC-1 TTP(#1) + 0.3 Experiment 2 Dermal MVEC-2 control — 0 Dermal MVEC-2 TTP (#2)— 20.0 Dermal MVEC-2 TTP (#2) control Ab 13.1 Dermal MVEC-2 TTP (#2) +0.2 Experiment 3 Dermal MVEC-1 TTP (#1) — 50.1 Dermal MVEC-1 TTP (#1) +10.6 Experiment 4 Dermal MVEC-2 control — 0 Dermal MVEC-2 TTP (#1) —13.9 Dermal MVEC-2 TTP (#1) control Ab 14.1 Dermal MVEC-2 TTP (#1) + 0.6

The data reveal that plasma derived from TTP patients induces apoptosisof microvascular endothelial cells of dermal origin. This apoptosis wasinhibited by polyclonal antibodies directed against TRAIL.

EXAMPLE 14

Expression of LZ-TRAIL

Examples of fusion proteins comprising leucine zipper (LZ) peptidesfused to the N-terminus of a soluble TRAIL polypeptide are as follows.The leucine zipper moieties promote oligomerization of the TRAILpolypeptides fused thereto, as described above.

An expression vector is constructed, containing DNA encoding (from N- toC-terminus) a human growth hormone signal peptide, a leucine zipperpeptide, and a soluble human TRAIL polypeptide. The TRAIL polypeptidecomprises amino acids 95 to 281 of SEQ ID NO:2. This TRAIL polypeptideis a fragment of the extracellular domain of human TRAL, lacking thespacer region, as described in example 7.

The growth hormone signal peptide comprises the amino acid sequence: MetAla Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu ProTrp Leu Gln Glu Gly Ser Ala (SEQ ID NO:19). The leucine zipper peptideis selected from peptides comprising the amino acid sequence:

(Asp)_(n) (Arg)_(n) Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu SerLys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly GluArg, wherein each n independently represents 1 or 0 (SEQ ID NO:15);

Ser Leu Ala Ser Leu Arg Gln Gln Leu Glu Ala Leu Gln Gly Gln Leu Gln HisLeu Gln Ala Ala Leu Ser Gln Leu Gly Glu (SEQ ID NO:16); or

Ser Ile Ala Ser Ile Arg Gln Gln Ile Glu Ala Ile Gln Gly Gln Ile Gln HisIle Gln Ala Ala Ile Ser Gln Ile Gly Glu (SEQ ID NO:17).

The fusion protein may comprise additional amino acid residue(s),encoded by DNA segments that result from construction of the vector, orthat are added to facilitate vector construction. In one particularembodiment, the tripeptide Thr-Ser-Ser is positioned between the growthhormone signal peptide and the leucine zipper peptide. This tripeptideis encoded by a DNA segment that comprises an Spe I restrictionendonuclease recognition site. The tripeptide Thr-Arg-Ser, encoded by aDNA segment that comprises a Bgl II restriction site, may be positionedbetween the leucine zipper and the TRAIL polypeptide.

DNA encoding the desired fusion protein is inserted into a suitableexpression vector, such as the pDC409 vector described in example 7.CV1-EBNA cells are transformed with the recombinant expression vector,then cultured to allow expression of the fusion protein, andoligomerization thereof.

25 1 1751 DNA human CDS (88)..(933) 1 cctcactgac tataaaagaa tagagaaggaagggcttcag tgaccggctg cctggctgac 60 ttacagcagt cagactctga caggatc atggct atg atg gag gtc cag ggg gga 114 Met Ala Met Met Glu Val Gln Gly Gly1 5 ccc agc ctg gga cag acc tgc gtg ctg atc gtg atc ttc aca gtg ctc 162Pro Ser Leu Gly Gln Thr Cys Val Leu Ile Val Ile Phe Thr Val Leu 10 15 2025 ctg cag tct ctc tgt gtg gct gta act tac gtg tac ttt acc aac gag 210Leu Gln Ser Leu Cys Val Ala Val Thr Tyr Val Tyr Phe Thr Asn Glu 30 35 40ctg aag cag atg cag gac aag tac tcc aaa agt ggc att gct tgt ttc 258 LeuLys Gln Met Gln Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys Phe 45 50 55 ttaaaa gaa gat gac agt tat tgg gac ccc aat gac gaa gag agt atg 306 Leu LysGlu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser Met 60 65 70 aac agcccc tgc tgg caa gtc aag tgg caa ctc cgt cag ctc gtt aga 354 Asn Ser ProCys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val Arg 75 80 85 aag atg attttg aga acc tct gag gaa acc att tct aca gtt caa gaa 402 Lys Met Ile LeuArg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu 90 95 100 105 aag caacaa aat att tct ccc cta gtg aga gaa aga ggt cct cag aga 450 Lys Gln GlnAsn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg 110 115 120 gta gcagct cac ata act ggg acc aga gga aga agc aac aca ttg tct 498 Val Ala AlaHis Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser 125 130 135 tct ccaaac tcc aag aat gaa aag gct ctg ggc cgc aaa ata aac tcc 546 Ser Pro AsnSer Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser 140 145 150 tgg gaatca tca agg agt ggg cat tca ttc ctg agc aac ttg cac ttg 594 Trp Glu SerSer Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu 155 160 165 agg aatggt gaa ctg gtc atc cat gaa aaa ggg ttt tac tac atc tat 642 Arg Asn GlyGlu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr 170 175 180 185 tcccaa aca tac ttt cga ttt cag gag gaa ata aaa gaa aac aca aag 690 Ser GlnThr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys 190 195 200 aacgac aaa caa atg gtc caa tat att tac aaa tac aca agt tat cct 738 Asn AspLys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro 205 210 215 gaccct ata ttg ttg atg aaa agt gct aga aat agt tgt tgg tct aaa 786 Asp ProIle Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys 220 225 230 gatgca gaa tat gga ctc tat tcc atc tat caa ggg gga ata ttt gag 834 Asp AlaGlu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu 235 240 245 cttaag gaa aat gac aga att ttt gtt tct gta aca aat gag cac ttg 882 Leu LysGlu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu 250 255 260 265ata gac atg gac cat gaa gcc agt ttt ttc ggg gcc ttt tta gtt ggc 930 IleAsp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 270 275 280taa ctgacctgga aagaaaaagc aataacctca aagtgactat tcagttttca 983ggatgataca ctatgaagat gtttcaaaaa atctgaccaa aacaaacaaa cagaaaacag 1043aaaacaaaaa aacctctatg caatctgagt agagcagcca caaccaaaaa attctacaac 1103acacactgtt ctgaaagtga ctcacttatc ccaagaaaat gaaattgctg aaagatcttt 1163caggactcta cctcatatca gtttgctagc agaaatctag aagactgtca gcttccaaac 1223attaatgcaa tggttaacat cttctgtctt tataatctac tccttgtaaa gactgtagaa 1283gaaagcgcaa caatccatct ctcaagtagt gtatcacagt agtagcctcc aggtttcctt 1343aagggacaac atccttaagt caaaagagag aagaggcacc actaaaagat cgcagtttgc 1403ctggtgcagt ggctcacacc tgtaatccca acattttggg aacccaaggt gggtagatca 1463cgagatcaag agatcaagac catagtgacc aacatagtga aaccccatct ctactgaaag 1523tgcaaaaatt agctgggtgt gttggcacat gcctgtagtc ccagctactt gagaggctga 1583ggcaggagaa tcgtttgaac ccgggaggca gaggttgcag tgtggtgaga tcatgccact 1643acactccagc ctggcgacag agcgagactt ggtttcaaaa aaaaaaaaaa aaaaaaactt 1703cagtaagtac gtgttatttt tttcaataaa attctattac agtatgtc 1751 2 281 PRThuman 2 Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys ValAla 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln AspLys 35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp SerTyr 50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp GlnVal 65 70 75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu ArgThr Ser 85 90 95 Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn IleSer Pro 100 105 110 Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala HisIle Thr Gly 115 120 125 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro AsnSer Lys Asn Glu 130 135 140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp GluSer Ser Arg Ser Gly 145 150 155 160 His Ser Phe Leu Ser Asn Leu His LeuArg Asn Gly Glu Leu Val Ile 165 170 175 His Glu Lys Gly Phe Tyr Tyr IleTyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190 Gln Glu Glu Ile Lys Glu AsnThr Lys Asn Asp Lys Gln Met Val Gln 195 200 205 Tyr Ile Tyr Lys Tyr ThrSer Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220 Ser Ala Arg Asn SerCys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240 Ser Ile TyrGln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 245 250 255 Phe ValSer Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 260 265 270 SerPhe Phe Gly Ala Phe Leu Val Gly 275 280 3 1521 DNA human CDS (78)..(383)3 aattccggaa tagagaagga agggcttcag tgaccggctg cctggctgac ttacagcagt 60cagactctga caggatc atg gct atg atg gag gtc cag ggg gga ccc agc 110 MetAla Met Met Glu Val Gln Gly Gly Pro Ser 1 5 10 ctg gga cag acc tgc gtgctg atc gtg atc ttc aca gtg ctc ctg cag 158 Leu Gly Gln Thr Cys Val LeuIle Val Ile Phe Thr Val Leu Leu Gln 15 20 25 tct ctc tgt gtg gct gta acttac gtg tac ttt acc aac gag ctg aag 206 Ser Leu Cys Val Ala Val Thr TyrVal Tyr Phe Thr Asn Glu Leu Lys 30 35 40 cag atg cag gac aag tac tcc aaaagt ggc att gct tgt ttc tta aaa 254 Gln Met Gln Asp Lys Tyr Ser Lys SerGly Ile Ala Cys Phe Leu Lys 45 50 55 gaa gat gac agt tat tgg gac ccc aatgac gaa gag agt atg aac agc 302 Glu Asp Asp Ser Tyr Trp Asp Pro Asn AspGlu Glu Ser Met Asn Ser 60 65 70 75 ccc tgc tgg caa gtc aag tgg caa ctccgt cag ctc gtt aga aag act 350 Pro Cys Trp Gln Val Lys Trp Gln Leu ArgGln Leu Val Arg Lys Thr 80 85 90 cca aga atg aaa agg ctc tgg gcc gca aaataa actcctggga atcatcaagg 403 Pro Arg Met Lys Arg Leu Trp Ala Ala Lys 95100 agtgggcatt cattcctgag caacttgcac ttgaggaatg gtgaactggt catccatgaa463 aaagggtttt actacatcta ttcccaaaca tactttcgat ttcaggagga aataaaagaa523 aacacaaaga acgacaaaca aatggtccaa tatatttaca aatacacaag ttatcctgac583 cctatattgt tgatgaaaag tgctagaaat agttgttggt ctaaagatgc agaatatgga643 ctctattcca tctatcaagg gggaatattt gagcttaagg aaaatgacag aatttttgtt703 tctgtaacaa atgagcactt gatagacatg gaccatgaag ccagtttttt cggggccttt763 ttagttggct aactgacctg gaaagaaaaa gcaataacct caaagtgact attcagtttt823 caggatgata cactatgaag atgtttcaaa aaatctgacc aaaacaaaca aacagaaaac883 agaaaacaaa aaaacctcta tgcaatctga gtagagcagc cacaaccaaa aaattctaca943 acacacactg ttctgaaagt gactcactta tcccaagaga atgaaattgc tgaaagatct1003 ttcaggactc tacctcatat cagtttgcta gcagaaatct agaagactgt cagcttccaa1063 acattaatgc agtggttaac atcttctgtc tttataatct actccttgta aagactgtag1123 aagaaagcgc aacaatccat ctctcaagta gtgtatcaca gtagtagcct ccaggtttcc1183 ttaagggaca acatccttaa gtcaaaagag agaagaggca ccactaaaag atcgcagttt1243 gcctggtgca gtggctcaca cctgtaatcc caacattttg ggaacccaag gtgggtagat1303 cacgagatca agagatcaag accatagtga ccaacatagt gaaaccccat ctctactgaa1363 agtgcaaaaa ttagctgggt gtgttggcac atgcctgtag tcccagctac ttgagaggct1423 gaggcaggag aatcgtttga acccgggagg cagaggttgc agtgtggtga gatcatgcca1483 ctacactcca gcctggcgac agagcgagac ttggtttc 1521 4 101 PRT human 4Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 1 5 1015 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 2530 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 4045 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 5560 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 65 7075 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Thr Pro Arg Met Lys Arg 8590 95 Leu Trp Ala Ala Lys 100 5 1366 DNA murine CDS (47)..(919) 5tgctgggctg caagtctgca ttgggaagtc agacctggac agcagt atg cct tcc 55 MetPro Ser 1 tca ggg gcc ctg aag gac ctc agc ttc agt cag cac ttc agg atgatg 103 Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gln His Phe Arg Met Met5 10 15 gtg att tgc ata gtg ctc ctg cag gtg ctc ctg cag gct gtg tct gtg151 Val Ile Cys Ile Val Leu Leu Gln Val Leu Leu Gln Ala Val Ser Val 2025 30 35 gct gtg act tac atg tac ttc acc aac gag atg aag cag ctg cag gac199 Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys Gln Leu Gln Asp 4045 50 aat tac tcc aaa att gga cta gct tgc ttc tca aag acg gat gag gat247 Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser Lys Thr Asp Glu Asp 5560 65 ttc tgg gac tcc act gat gga gag atc ttg aac aga ccc tgc ttg cag295 Phe Trp Asp Ser Thr Asp Gly Glu Ile Leu Asn Arg Pro Cys Leu Gln 7075 80 gtt aag agg caa ctg tat cag ctc att gaa gag gtg act ttg aga acc343 Val Lys Arg Gln Leu Tyr Gln Leu Ile Glu Glu Val Thr Leu Arg Thr 8590 95 ttt cag gac acc att tct aca gtt cca gaa aag cag cta agt act cct391 Phe Gln Asp Thr Ile Ser Thr Val Pro Glu Lys Gln Leu Ser Thr Pro 100105 110 115 ccc ttg ccc aga ggt gga aga cct cag aaa gtg gca gct cac attact 439 Pro Leu Pro Arg Gly Gly Arg Pro Gln Lys Val Ala Ala His Ile Thr120 125 130 ggg atc act cgg aga agc aac tca gct tta att cca atc tcc aaggat 487 Gly Ile Thr Arg Arg Ser Asn Ser Ala Leu Ile Pro Ile Ser Lys Asp135 140 145 gga aag acc tta ggc cag aag att gaa tcc tgg gag tcc tct cggaaa 535 Gly Lys Thr Leu Gly Gln Lys Ile Glu Ser Trp Glu Ser Ser Arg Lys150 155 160 ggg cat tca ttt ctc aac cac gtg ctc ttt agg aat gga gag ctggtc 583 Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn Gly Glu Leu Val165 170 175 atc gag cag gag ggc ctg tat tac atc tat tcc caa aca tac ttccga 631 Ile Glu Gln Glu Gly Leu Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg180 185 190 195 ttt cag gaa gct gaa gac gct tcc aag atg gtc tca aag gacaag gtg 679 Phe Gln Glu Ala Glu Asp Ala Ser Lys Met Val Ser Lys Asp LysVal 200 205 210 aga acc aaa cag ctg gtg cag tac atc tac aag tac acc agctat ccg 727 Arg Thr Lys Gln Leu Val Gln Tyr Ile Tyr Lys Tyr Thr Ser TyrPro 215 220 225 gat ccc ata gtg ctc atg aag agc gcc aga aac agc tgt tggtcc aga 775 Asp Pro Ile Val Leu Met Lys Ser Ala Arg Asn Ser Cys Trp SerArg 230 235 240 gat gcc gag tac gga ctg tac tcc atc tat cag gga gga ttgttc gag 823 Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Leu PheGlu 245 250 255 cta aaa aaa aat gac agg att ttt gtt tct gtg aca aat gaacat ttg 871 Leu Lys Lys Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu HisLeu 260 265 270 275 atg gac ctg gat caa gaa gcc agc ttc ttt gga gcc ttttta att aac 919 Met Asp Leu Asp Gln Glu Ala Ser Phe Phe Gly Ala Phe LeuIle Asn 280 285 290 taaatgacca gtaaagatca aacacagccc taaagtacccagtaatcttc taggttgaag 979 gcatgcctgg aaagcgactg aactggttag gatatggcctggctgtagaa acctcaggac 1039 agatgtgaca gaaaggcagc tggaactcag cagcgacaggccaacagtcc agccacagac 1099 actttcggtg tttcatcgag agacttgctt tctttccgcaaaatgagatc actgtagcct 1159 ttcaatgatc tacctggtat cagtttgcag agatctagaagacgtccagt ttctaaatat 1219 ttatgcaaca attgacaatt ttcacctttg ttatctggtccaggggtgta aagccaagtg 1279 ctcacaagct gtgtgcagac caggatagct atgaatgcaggtcagcataa aaatcacaga 1339 atatctcacc tactaaaaaa aaaaaaa 1366 6 291 PRTmurine 6 Met Pro Ser Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gln His Phe1 5 10 15 Arg Met Met Val Ile Cys Ile Val Leu Leu Gln Val Leu Leu GlnAla 20 25 30 Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met LysGln 35 40 45 Leu Gln Asp Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser LysThr 50 55 60 Asp Glu Asp Phe Trp Asp Ser Thr Asp Gly Glu Ile Leu Asn ArgPro 65 70 75 80 Cys Leu Gln Val Lys Arg Gln Leu Tyr Gln Leu Ile Glu GluVal Thr 85 90 95 Leu Arg Thr Phe Gln Asp Thr Ile Ser Thr Val Pro Glu LysGln Leu 100 105 110 Ser Thr Pro Pro Leu Pro Arg Gly Gly Arg Pro Gln LysVal Ala Ala 115 120 125 His Ile Thr Gly Ile Thr Arg Arg Ser Asn Ser AlaLeu Ile Pro Ile 130 135 140 Ser Lys Asp Gly Lys Thr Leu Gly Gln Lys IleGlu Ser Trp Glu Ser 145 150 155 160 Ser Arg Lys Gly His Ser Phe Leu AsnHis Val Leu Phe Arg Asn Gly 165 170 175 Glu Leu Val Ile Glu Gln Glu GlyLeu Tyr Tyr Ile Tyr Ser Gln Thr 180 185 190 Tyr Phe Arg Phe Gln Glu AlaGlu Asp Ala Ser Lys Met Val Ser Lys 195 200 205 Asp Lys Val Arg Thr LysGln Leu Val Gln Tyr Ile Tyr Lys Tyr Thr 210 215 220 Ser Tyr Pro Asp ProIle Val Leu Met Lys Ser Ala Arg Asn Ser Cys 225 230 235 240 Trp Ser ArgAsp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly 245 250 255 Leu PheGlu Leu Lys Lys Asn Asp Arg Ile Phe Val Ser Val Thr Asn 260 265 270 GluHis Leu Met Asp Leu Asp Gln Glu Ala Ser Phe Phe Gly Ala Phe 275 280 285Leu Ile Asn 290 7 8 PRT synthetic 7 Asp Tyr Lys Asp Asp Asp Asp Lys 1 58 17 PRT conserved peptide 8 Leu Val Val Xaa Xaa Xaa Gly Leu Tyr Tyr ValTyr Xaa Gln Val Xaa 1 5 10 15 Phe 9 32 PRT CMV leader 9 Met Ala Arg ArgLeu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 15 Val Ala LeuAla Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser Ser 20 25 30 10 759 DNAsynthetic fusion CDS (1)..(759) 10 atg gct aca ggc tcc cgg acg tcc ctgctc ctg gct ttt ggc ctg ctc 48 Met Ala Thr Gly Ser Arg Thr Ser Leu LeuLeu Ala Phe Gly Leu Leu 1 5 10 15 tgc ctg ccc tgg ctt caa gag ggc agtgca act agt tct gac cgt atg 96 Cys Leu Pro Trp Leu Gln Glu Gly Ser AlaThr Ser Ser Asp Arg Met 20 25 30 aaa cag ata gag gat aag atc gaa gag atccta agt aag att tat cat 144 Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile LeuSer Lys Ile Tyr His 35 40 45 ata gag aat gaa atc gcc cgt atc aaa aag ctgatt ggc gag cgg act 192 Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu IleGly Glu Arg Thr 50 55 60 aga tct acc tct gag gaa acc att tct aca gtt caagaa aag caa caa 240 Arg Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln GluLys Gln Gln 65 70 75 80 aat att tct ccc cta gtg aga gaa aga ggt cct cagaga gta gca gct 288 Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln ArgVal Ala Ala 85 90 95 cac ata act ggg acc aga gga aga agc aac aca ttg tcttct cca aac 336 His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser SerPro Asn 100 105 110 tcc aag aat gaa aag gct ctg ggc cgc aaa ata aac tcctgg gaa tca 384 Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser TrpGlu Ser 115 120 125 tca agg agt ggg cat tca ttc ctg agc aac ttg cac ttgagg aat ggt 432 Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu ArgAsn Gly 130 135 140 gaa ctg gtc atc cat gaa aaa ggg ttt tac tac atc tattcc caa aca 480 Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr SerGln Thr 145 150 155 160 tac ttt cga ttt cag gag gaa ata aaa gaa aac acaaag aac gac aaa 528 Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr LysAsn Asp Lys 165 170 175 caa atg gtc caa tat att tac aaa tac aca agt tatcct gac cct ata 576 Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr ProAsp Pro Ile 180 185 190 ttg ttg atg aaa agt gct aga aat agt tgt tgg tctaaa gat gca gaa 624 Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser LysAsp Ala Glu 195 200 205 tat gga ctc tat tcc atc tat caa ggg gga ata tttgag ctt aag gaa 672 Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe GluLeu Lys Glu 210 215 220 aat gac aga att ttt gtt tct gta aca aat gag cacttg ata gac atg 720 Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His LeuIle Asp Met 225 230 235 240 gac cat gaa gcc agt ttt ttc ggg gcc ttt ttagtt ggc 759 Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245 25011 253 PRT synthetic fusion 11 Met Ala Thr Gly Ser Arg Thr Ser Leu LeuLeu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly SerAla Thr Ser Ser Asp Arg Met 20 25 30 Lys Gln Ile Glu Asp Lys Ile Glu GluIle Leu Ser Lys Ile Tyr His 35 40 45 Ile Glu Asn Glu Ile Ala Arg Ile LysLys Leu Ile Gly Glu Arg Thr 50 55 60 Arg Ser Thr Ser Glu Glu Thr Ile SerThr Val Gln Glu Lys Gln Gln 65 70 75 80 Asn Ile Ser Pro Leu Val Arg GluArg Gly Pro Gln Arg Val Ala Ala 85 90 95 His Ile Thr Gly Thr Arg Gly ArgSer Asn Thr Leu Ser Ser Pro Asn 100 105 110 Ser Lys Asn Glu Lys Ala LeuGly Arg Lys Ile Asn Ser Trp Glu Ser 115 120 125 Ser Arg Ser Gly His SerPhe Leu Ser Asn Leu His Leu Arg Asn Gly 130 135 140 Glu Leu Val Ile HisGlu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr 145 150 155 160 Tyr Phe ArgPhe Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys 165 170 175 Gln MetVal Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile 180 185 190 LeuLeu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu 195 200 205Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu 210 215220 Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met 225230 235 240 Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245 25012 768 DNA synthetic fusion CDS (1)..(768) 12 atg gct cgg agg cta tggatc ttg agc tta tta gcc gtg acc ttg acg 48 Met Ala Arg Arg Leu Trp IleLeu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 15 gtg gct ttg gcg gca ccttct cag aaa tcg aag cgc agg act agt tct 96 Val Ala Leu Ala Ala Pro SerGln Lys Ser Lys Arg Arg Thr Ser Ser 20 25 30 gac cgt atg aaa cag ata gaggat aag atc gaa gag atc cta agt aag 144 Asp Arg Met Lys Gln Ile Glu AspLys Ile Glu Glu Ile Leu Ser Lys 35 40 45 att tat cat ata gag aat gaa atcgcc cgt atc aaa aag ctg att ggc 192 Ile Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly 50 55 60 gag cgg act aga tct acc tct gag gaaacc att tct aca gtt caa gaa 240 Glu Arg Thr Arg Ser Thr Ser Glu Glu ThrIle Ser Thr Val Gln Glu 65 70 75 80 aag caa caa aat att tct ccc cta gtgaga gaa aga ggt cct cag aga 288 Lys Gln Gln Asn Ile Ser Pro Leu Val ArgGlu Arg Gly Pro Gln Arg 85 90 95 gta gca gct cac ata act ggg acc aga ggaaga agc aac aca ttg tct 336 Val Ala Ala His Ile Thr Gly Thr Arg Gly ArgSer Asn Thr Leu Ser 100 105 110 tct cca aac tcc aag aat gaa aag gct ctgggc cgc aaa ata aac tcc 384 Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu GlyArg Lys Ile Asn Ser 115 120 125 tgg gaa tca tca agg agt ggg cat tca ttcctg agc aac ttg cac ttg 432 Trp Glu Ser Ser Arg Ser Gly His Ser Phe LeuSer Asn Leu His Leu 130 135 140 agg aat ggt gaa ctg gtc atc cat gaa aaaggg ttt tac tac atc tat 480 Arg Asn Gly Glu Leu Val Ile His Glu Lys GlyPhe Tyr Tyr Ile Tyr 145 150 155 160 tcc caa aca tac ttt cga ttt cag gaggaa ata aaa gaa aac aca aag 528 Ser Gln Thr Tyr Phe Arg Phe Gln Glu GluIle Lys Glu Asn Thr Lys 165 170 175 aac gac aaa caa atg gtc caa tat atttac aaa tac aca agt tat cct 576 Asn Asp Lys Gln Met Val Gln Tyr Ile TyrLys Tyr Thr Ser Tyr Pro 180 185 190 gac cct ata ttg ttg atg aaa agt gctaga aat agt tgt tgg tct aaa 624 Asp Pro Ile Leu Leu Met Lys Ser Ala ArgAsn Ser Cys Trp Ser Lys 195 200 205 gat gca gaa tat gga ctc tat tcc atctat caa ggg gga ata ttt gag 672 Asp Ala Glu Tyr Gly Leu Tyr Ser Ile TyrGln Gly Gly Ile Phe Glu 210 215 220 ctt aag gaa aat gac aga att ttt gtttct gta aca aat gag cac ttg 720 Leu Lys Glu Asn Asp Arg Ile Phe Val SerVal Thr Asn Glu His Leu 225 230 235 240 ata gac atg gac cat gaa gcc agtttt ttc ggg gcc ttt tta gtt ggc 768 Ile Asp Met Asp His Glu Ala Ser PhePhe Gly Ala Phe Leu Val Gly 245 250 255 13 256 PRT synthetic fusion 13Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 1015 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser Ser 20 2530 Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 35 4045 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 50 5560 Glu Arg Thr Arg Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu 65 7075 80 Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg 8590 95 Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser100 105 110 Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile AsnSer 115 120 125 Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn LeuHis Leu 130 135 140 Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe TyrTyr Ile Tyr 145 150 155 160 Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu IleLys Glu Asn Thr Lys 165 170 175 Asn Asp Lys Gln Met Val Gln Tyr Ile TyrLys Tyr Thr Ser Tyr Pro 180 185 190 Asp Pro Ile Leu Leu Met Lys Ser AlaArg Asn Ser Cys Trp Ser Lys 195 200 205 Asp Ala Glu Tyr Gly Leu Tyr SerIle Tyr Gln Gly Gly Ile Phe Glu 210 215 220 Leu Lys Glu Asn Asp Arg IlePhe Val Ser Val Thr Asn Glu His Leu 225 230 235 240 Ile Asp Met Asp HisGlu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245 250 255 14 27 PRT LZpeptide 14 Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln GlyGln 1 5 10 15 Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr 20 25 15 34PRT LZ peptide 15 Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu IleLeu Ser Lys 1 5 10 15 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile LysLys Leu Ile Gly 20 25 30 Glu Arg 16 28 PRT LZ peptide 16 Ser Leu Ala SerLeu Arg Gln Gln Leu Glu Ala Leu Gln Gly Gln Leu 1 5 10 15 Gln His LeuGln Ala Ala Leu Ser Gln Leu Gly Glu 20 25 17 28 PRT LZ peptide 17 SerIle Ala Ser Ile Arg Gln Gln Ile Glu Ala Ile Gln Gly Gln Ile 1 5 10 15Gln His Ile Gln Ala Ala Ile Ser Gln Ile Gly Glu 20 25 18 77 DNA GHLeader 18 atggctacag gctcccggac gtccctgtcc tggcttttgg cctgctctgcctgccctggc 60 ttcaagaggg cagtgca 77 19 26 PRT GH Leader 19 Met Ala ThrGly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys LeuPro Trp Leu Gln Glu Gly Ser Ala 20 25 20 9 PRT Artificial SequenceDescription of Artificial Sequence artificial peptide 20 Cys Asp Cys ArgGly Asp Cys Phe Cys 1 5 21 13 PRT Artificial Sequence Description ofArtificial Sequence artificial peptide 21 Cys Asn Gly Arg Cys Val SerGly Cys Ala Gly Arg Cys 1 5 10 22 6 PRT Artificial Sequence Descriptionof Artificial Sequence artificial peptide 22 Asn Gly Arg Ala His Ala 1 523 9 PRT Artificial Sequence Description of Artificial Sequenceartificial peptide 23 Cys Val Leu Asn Gly Arg Met Glu Cys 1 5 24 5 PRTArtificial Sequence Description of Artificial Sequence artificialpeptide 24 Cys Asn Gly Arg Cys 1 5 25 20 PRT Homo sapiens 25 Met Gly ThrAsp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly SerThr Gly 20

What is claimed is:
 1. An antibody that specifically binds the humantumor necrosis factor related apoptosis inducing ligand (TRAIL) proteinof SEQ ID NO:2.
 2. An antibody according to claim 1, wherein saidantibody is a monoclonal antibody.
 3. An antibody of claim 1, whereinthe antibody inhibits TRAIL-mediated apoptosis of a target cell.
 4. Anantibody of claim 1, wherein the antibody blocks binding of TRAIL to atarget cell.
 5. An antibody of claim 4, wherein said antibody is amonoclonal antibody.
 6. An antigen-binding fragment of an antibody ofclaim
 1. 7. An antigen-binding fragment of a monoclonal antibody ofclaim
 5. 8. A hybridoma cell line that produces a monoclonal antibody ofclaim
 2. 9. A composition comprising an antibody of claim 1, and aphysiologically acceptable carrier, diluent, or excipient.
 10. Acomposition comprising a monoclonal antibody of claim 2, and aphysiologically acceptable carrier, diluent, or excipient.
 11. Anantibody of claim 3, wherein said antibody is an monoclonal antibody.12. An antigen-binding fragment of a monoclonal antibody of claim 11.13. An antibody that specifically binds a soluble human TRAILpolypeptide, wherein said polypeptide comprises amino acids x to 281 ofSEQ ID NO:2, wherein x represents an integer from 39 to
 95. 14. Anantibody of claim 13, wherein said antibody is a monoclonal antibody.15. An antibody of claim 13, wherein the antibody inhibitsTRAIL-mediated apoptosis of a target cell.
 16. A monoclonal antibody ofclaim 14, wherein the antibody blocks binding of TRAIL to a target cell.17. An antigen-binding fragment of a monoclonal antibody of claim 14.18. A hybridoma cell line that produces a monoclonal antibody of claim14.
 19. An antibody of claim 15, wherein said antibody is a monoclonalantibody.
 20. An antibody that binds a polypeptide comprising aminoacids 124 to 276 of SEQ ID NO:2.
 21. An antibody of claim 20, whereinsaid antibody is a monoclonal antibody.
 22. An antibody of claim 20,wherein the antibody inhibits TRAIL-mediated apoptosis of a target cell.23. A monoclonal antibody of claim 21, wherein the antibody blocksbinding of TRAIL to a target cell.
 24. An antigen-binding fragment of amonoclonal antibody of claim
 21. 25. An antibody of claim 22, whereinsaid antibody is a monoclonal antibody.
 26. An antibody that binds afragment of the TRAIL protein of SEQ ID NO:2, wherein the N-terminalamino acid of said fragment is selected from residues 39 to 124 of SEQID NO:2, and the C-terminal amino acid of said fragment is selected fromresidues 276 to 281 of SEQ ID NO:2.
 27. An antibody of claim 26, whereinsaid antibody is a monoclonal antibody.
 28. An antibody of claim 26,wherein the antibody inhibits TRAIL-mediated apoptosis of a target cell.29. A monoclonal antibody of claim 27 wherein the antibody blocksbinding of TRAIL to a target cell.
 30. An antigen-binding fragment of amonoclonal antibody of claim
 27. 31. A hybridoma cell line that producesa monoclonal antibody of claim
 27. 32. A hybridoma cell line thatproduces a monoclonal antibody of claim
 29. 33. An antibody of claim 28,wherein said antibody is a monoclonal antibody.
 34. An antigen-bindingfragment of a monoclonal antibody of claim
 33. 35. A hybridoma cell linethat produces a monoclonal antibody of claim 33.